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  • 98
    Millipore mouse monoclonal antibody against epha2
    Reciprocal regulation of ephrin-A1 and <t>EphA2</t> expression in corneal epithelial cell cultures. (A) Immunostaining of EphA2 (green), ephrin-A1 (red), and E-cadherin (magenta) in mono-cultures of hTCEPi cells transduced with an empty control or an ephrin-A1 cDNA construct. Representative images from n = 3. (B) Immunoblotting for total EphA2, ephrin-A1, or E-cadherin in hTCEPi cells overexpressing ephrin-A1 (EFNA1). GAPDH was used as a protein loading control. Representative blots from n = 4. (C) Immunostaining of EphA2 (green), ephrin-A1 (red), and E-cadherin (magenta) in hTCEPi cells knocked down for ephrin-A1 (siEphrin-A1), EphA2 (siEphA2), or both proteins (siEphA2+siEphrin-A1). Scale bar denotes 100 μm. Representative images from n = 3. (D) Immunoblotting for total EphA2, ephrin-A1, or E-cadherin in hTCEPi cells with siRNA targeted knockdown of ephrin-A1, EphA2, or double knockdown. GAPDH was used as a protein loading control. Representative blots from n = 3.
    Mouse Monoclonal Antibody Against Epha2, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    RayBiotech p75ntr ngfr rabbit polyclonal antibody
    The <t>NGFR</t> High phenotype can be suppressed by JQ 1 in BRAF V 600E melanoma xenografts Immunofluorescence analysis of A375 melanoma xenograft tumors co‐stained for Ki‐67 and NGFR proteins. Selected images from a whole tumor section as well as NGFR High /Ki‐67 Low and NGFR Low /Ki‐67 High regions of a vehicle‐treated tumor are shown to highlight the spatial and cell‐to‐cell heterogeneity in Ki‐67 and NGFR protein expression. Percentage of Ki‐67 High and NGFR High cells in tumors treated for 5 days with dabrafenib (25 mg/kg) only, dabrafenib (25 mg/kg) in combination with JQ1 (50 mg/kg), or vehicle. Number of tumors (mice) analyzed per condition is shown. Solid horizontal lines represent the mean of measurements. Up to 50,000 individual cells per tumor were analyzed for NGFR and Ki‐67 intensities. Statistical significance was determined using two‐tailed two‐sample t ‐test.
    P75ntr Ngfr Rabbit Polyclonal Antibody, supplied by RayBiotech, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti α tubulin antibody
    The CPC and CENP-E exhibit distinct central spindle transfer mechanisms during anaphase. (A–F) CENP-E transfers to the central spindle at anaphase even when the CPC remains stuck on the chromatin. CENP-E (green; panels 3 and 7) and <t>α-tubulin</t> or TrAP-INCENP (red; panels 2 and 6) plus DAPI for DNA (blue; panels 4 and 8) are shown for INCENP ON cells (A), INCENP OFF cells (B), or INCENP OFF cells expressing TrAP-INCENP WT (C), TrAP-INCENP W766G (D), TrAP-INCENP F802A (E), or TrAP-INCENP WT in the presence of ZM447439 (F). CENP-E localizes to the outer kinetochore in all cells (insets) and transfers to the central spindle at anaphase, even in cells expressing TrAP-INCENP W766G or TrAP-INCENP F802A in which the CPC remains trapped on the chromatin. Insets show magnified views of boxed regions. Bars, 5 µm. (G) The level of H3S10ph is reduced significantly in the presence of ZM447439 as a result of aurora B inhibition. Levels of aurora B and PP1 remained the same. α-Tubulin was used as a loading control.
    Anti α Tubulin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1727 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore cofilin
    A model for defective huntingtin-mediated <t>cofilin</t> rod stress response leading to activation of TG2. The grey arrow pathway highlights normal huntingtin stress response by releasing from the ER, entering the nucleus and binding cofilin–actin rods, then exiting the nucleus upon stress relief. With mutant huntingtin present, the dashed arrow pathways show a defect in huntingtin stress response, resulting in less nuclear activity and persistent rods. Back in the cytoplasm, the black arrow pathways highlight elevated calcium due to a defective ER, which results in aberrant TG2 activation and cross-linking of cofilin–actin in both the nucleus and cytoplasm. Defective actin remodeling critically affects neurons at the level of dendritic and synaptic dysfunction, as well as exocytosis activity in peripheral cells.
    Cofilin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 82 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Millipore anti atg13
    COPI complex promotes elongation of autophagosomes. ( a ) HEK293 cells stably expressing GFP-DFCP1 or the parental cell line were pre-treated for 3 h with 3 μg ml −1 BFA (long BFA), starved for 1 h in the presence of BFA and the parental cell line was immunolabelled for WIPI2 or ATG16. Cells were imaged by wide-field microscopy. Short BFA corresponds to non-pre-treated cells. Bar corresponds to 10 μm. ( b ) Values are means±s.e.m. of DFCP1, WIPI2 and ATG16 spots per cell in a , from at least six fields with 5–10 cells each. ( c ) HEK293 cells were transfected with δCOP (siδCOP), β'COP (siβ'COP) or non-targeted (siNT) siRNA, starved, immunolabelled for WIPI2 and imaged by wide-field microscopy. Arrows point at WIPI2 ring-like structures. Bar corresponds to 10 μm. ( d ) Values are means±s.e.m. of WIPI2 rings per cell in c , for 10 different fields with 5–15 cells each. ( e ) HEK293 cells were pre-treated for 3 h with 3 μg ml −1 BFA, starved, immunolabelled for WIPI2 and imaged by wide-field microscopy. Values are means±s.e.m. of WIPI2 rings per cell, for 10 different fields with 5–15 cells each. ( f , g ) HEK293 cells stably expressing <t>GFP-ATG13</t> ( f ) or GFP-DFCP1 ( g ) were pre-treated for 3 h with 3 μg ml −1 BFA, starved and live-imaged in the presence of BFA by wide-field microscopy. The lifespan of ATG13 and DFCP1 particles was quantitated. Values are means±s.e.m. of ATG13 or DFCP1 particle lifespan, from 60 and 30 montages, respectively. ( h , i ) HEK293 cells expressing stably GFP-ATG13 ( h ) or GFP-DFCP1 ( i ) and transiently CFP-LC3 were pre-treated for 3 h with 3 μg ml −1 BFA, starved and live-imaged in the presence of BFA by wide-field microscopy. The lifespan of ATG13 and DFCP1 particles before the appearance of LC3 was quantitated. Values are means±s.e.m. of ATG13 or DFCP1 particle lifespan before the appearance of LC3, from 20 and 26 montages, respectively. Significance levels were determined with unpaired t -tests. * P =0.05%; ** P =0.01%; *** P =0.001%; **** P =0.0001%.
    Anti Atg13, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore rabbit anti vamp4
    Impact of siRNA knockdown on the localization of syntaxin 6 or <t>VAMP4</t> to the chlamydial inclusion. HeLa cells were treated with control (nontargeting), syntaxin 6, or VAMP4 siRNA and infected with C. trachomatis ( Ct ) for 18 h. Then, cells were fixed and
    Rabbit Anti Vamp4, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    BioLegend rat anti cd4
    <t>CD4</t> T cells home to the choroid plexus (CP) in an activation-, chemokine-, and intercellular adhesion molecule 1 (ICAM-1)-dependant manner. Intact lateral ventricle (LV) CPs from non-perfused CD45.2 mice, with or without lipopolysaccharide (LPS) preconditioning, were cocultured ex vivo with CD45.1 CD4 T cells in artificial CSF (aCSF) and then analyzed with flow cytometry and confocal microscopy. (A–E) Ex vivo cocultures, showing the homing of activated T cells to the CP with flow cytometry and live-cell imaging, and their impact on gene expression. (A) Flow cytometry analysis of the FSC-SSC T-cell population in untreated (UT) CPs cocultured for 24 h with CD45.1 + carboxyfluorescein succinimidyl ester-labeled non-activated (Non-activated CD4 + ; n = 4) or activated (Activated Th1; n = 4) T cells. (B,C) Confocal live-cell imaging of UT, GFP-labeled CPs, which were derived from UBC-GFP mice (B) and cocultured with SNARF-1 + Th1 cells adhering to and migrating within the CP [ (C) ; yellow arrows indicate interactions]. Scale bars represent 500 µm (B) , 20 µm (C) . (D) Homing kinetics of activated Th1 cells to the CP, after 1, 2, 5, and 24 h ( n = 5 at each time point) of ex vivo coculturing with untreated CPs. (E) A heat-map representation (fold change from control aCSF) of a quantitative PCR analysis of genes encoding immune mediators, performed on total RNA isolated from LV CPs that were cocultured with either non-activated CD4 + ( n = 4) or activated ( n = 4) Th1 cells, as compared with an aCSF control ( n = 3), 4 h after initiating the coculture. Fold changes and P values are provided in Tables S3A,B in Supplementary Material. (F,G) The role of chemokines and cell adhesion molecules in T-cell homing to the CP. (F) LV CPs were isolated from mice with or without LPS preconditioning, and they were then cocultured for 24 h with CD45.1 activated Th1 cells, either with a prior pre-treatment of the chemokine-signaling inhibitor pertussis toxin (PTX) ( n = 5 for LPS-preconditioned and n = 5 for phosphate-buffered saline-injected mice, respectively), or without it ( n = 5 and n = 4, respectively). Then, the CPs were analyzed by flow cytometry. The graph shows the frequency of CP-homing T cells. (G) CPs were isolated from LPS-preconditioned mice and then cultured for 24 h with CD45.1 activated Th1 cells in the presence of ICAM-1 neutralizing antibodies, or an isotype control ( n = 5 in each group). The graph shows the frequency of CP-homing T cells. Each symbol represents one LV CP from an individual mouse. Bars represent means ± SEM. * P
    Rat Anti Cd4, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher array scan hcs reader
    Increased expression of growth factor receptors (VEGFR1, FGFR2,) in lung CSCs. H460 cells and lung CSCs dissociated from spheres were plated into 96-well plates precoated with Collagen IV and cultured 8 h. Then adherent cells were incubated with FITC-conjugated Abs against FGFR2, VEGFR1 and VEGFR2 fixed and stained with Hoechst 33342. Images were acquired using the <t>Cellomics</t> ArrayScan <t>HCS</t> Reader (20X objective) and analyzed using the Target Activation BioApplication Software Module. A, Immunofluorescent images of VEGFR1 and FGFR2 in H460 and CSCs cells (20X objective). B, Fluorescence intensity (pix) of VEGFR1 and FGFR2 plotted against object area. Each point represents a single cell. In figures 8 – 10 red lines show the boundaries of the fluorescence intensity of H460 cells.
    Array Scan Hcs Reader, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore rabbit anti g3bp1
    <t>G3BP1</t> acidic domain increases axonal mRNA translation and disassembles stress granules. a , b Representative images for puromycin (Puro) incorporation in DRG neurons transfected with the indicated constructs are shown ( a ). Significant increase in axonal puromycin signals in the G3BP1 B domain-expressing neurons is seen, with no significant change in the cell body puromycin incorporation ( b ; N ≥ 23 axons over three repetitions; ** p ≤ 0.01, **** p ≤ 0.0001 by one-way ANOVA with Tukey HSD posthoc) [scale bar = 5 μm]. c G3BP1 depleted DRG cultures similarly show increased puromycin incorporation in axons with no significant change in cell body puromycin incorporation ( N ≥ 23 axons over three repetitions; ** p ≤ 0.01, **** p ≤ 0.0001 by one-way ANOVA with Tukey HSD posthoc). See Supplementary Fig. 7a for representative images of these puromycin incorporation studies. d Quantitation of endogenous axonal NRN1, IMPβ1, and GAP43 protein levels in DRG cultures transfected with GFP, G3BP1-GFP, and G3BP1 B domain-GFP is shown. Axonal NRN1 and IMPβ1 but not GAP43 levels are significantly reduced in G3BP1 overexpression. G3BP1 B domain-expressing neurons show significantly higher axonal NRN1, but no change in axonal IMPβ1 and GAP43 levels ( N ≥ 33 axons over three repetitions; * p ≤ 0.05, **** p ≤ 0.0001 by one-way ANOVA with Tukey HSD posthoc). Representative images for axonal immunofluorescence and cell body NRN1, IMPβ1, and GAP43 proteins are shown in Supplementary Fig. 7b, c . e RTddPCR for axonal mRNAs co-precipitating with G3BP1-GFP in DRG neurons are shown as average % mRNA associated with G3BP1-GFP ± SEM. Nrn1 and Impβ1 mRNAs association with G3BP1-GFP significantly reduced by cotransfection with the G3BP1 B domain, but neither RNA coprecipitates with the B domain ( N = 4 culture preparations; * p ≤ 0.05, ** p ≤ 0.01 by Student’s t -test for the indicated data pairs)
    Rabbit Anti G3bp1, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore anti ccdc170 antibody
    <t>CCDC170</t> protein levels affect the rate of 2D cell migration. a. Live cell imaging was carried out to monitor migratory activity of MCF-7 cells expressing either GFP-CCDC170 or non-fused GFP as a negative control. Cell migration tracks (blue) were quantified with ImageJ software by tracing the center of the cells. b. Average migration distance between GFP vs. GFP-CCDC170 overexpressing cells were compared ( t -test, * P
    Anti Ccdc170 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore rabbit anti ckap2
    <t>CKAP2</t> plays a role in maintaining chromosome stability. (A–C) Mitotic cells were treated with colcemid in order to obtain metaphase spreads. Chromosome content was determined by counting the individual chromosomes in at least 100 metaphases. The results are presented as radial plots, where the concentric circle represents the relative ploidy and each symbol represents an individual cell. In parallel, here indicated are karyotypes analyzed by SKY showing the increased level of aneuploidy and chromosome instability in CKAP2-depleted cells.
    Rabbit Anti Ckap2, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rabbit anti human pcsk9
    Live-cell imaging of WT <t>PCSK9,</t> PCSK9-ΔCTD-mC and LDLR-EGFP trafficking. HepG2 cells were co-transfected with LDLR-EGFP together with (A) WT PCSK9-mC (see S3 Video ) or (B) WT PCSK9-ΔCTD (see S4 Video ). Images were extracted from confocal microscopy time-lapse movies. Data are representative of at least three independent experiments. Scale bar , 20 μm.
    Rabbit Anti Human Pcsk9, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Millipore monoclonal anti dbn1 antibody
    SR-SIM reveals that f-actin and <t>drebrin</t> form a cortical collar around microtubules in the proximal leading process. ( a , b ) SR-SIM imaging of CGNs with dilated proximal leading processes expressing ( a ) GPI-pHluorin (green), drebrin E 2x-KO1 (red) and SiR-tubulin (blue; n =21 cells analysed) or ( b ) EGFP-UTRCH (green), Drebrin E 2x-KO1 (red) and SiR-tubulin (blue; n =18 cells analysed). Scale bar, 1 μm ( a ). The RGB plot to the right of each representative image shows the well-resolved Gaussian FWHM peaks detected for the line scan in each image (dashed white line). The box plots at far right show the measured distances between the centroid positions of the Gaussian peak for each fluorescent probe. Whiskers on the box plot show maximum and minimum data points, box borders show first and third quartiles and the line in the box show the median. ( c ) Drebrin is an f-actin and +TIP binding protein. Consistent with this interaction, LLS microscopy shows that microtubule +TIPs (labelled with EB3-2x Venus, green) pass through the drebrin E-labelled domain (labelled with drebrin E 2x-KO1, red) in the proximal leading process. Scale bar, 2μm ( c ). Time stamp=min:sec.
    Monoclonal Anti Dbn1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore mouse anti lck
    <t>RACK1–Lck</t> complexes can form irrespective of Lck kinase activity, but those from activated T-cells contain a sizeable fraction of pY394 Lck . (A) CD4 + T-cells precoated or non-precoated with biotinylated anti-TCR and anti-CD4 mAbs (TCRβ/CD4) were co-aggregated, or not (0 s), with streptavidin for the indicated period of time. RACK1 immunoprecipitates were blotted against pY394 Lck , total Lck, and RACK1. (B,C) Statistical analysis of pY394Lck and total Lck blots from (A) , respectively, represent the normalized to total RACK1 from at least three independent experiments. The statistical analysis presented as mean ± SD was performed using the Student’s two-tailed t -test, * p
    Mouse Anti Lck, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Millipore antibody against rab10
    Model for the <t>Rab10-EHBP1-EHD2</t> complex in mediating the autophagic engulfment of an LD during lipophagy. Following Rab7-mediated recruitment of degradative machinery to the LD surface, Rab10 works in a complex together with EHD2 and EHBP1 to promote extension of an LC3-positive autophagic membrane along the circumference of the LD surface.
    Antibody Against Rab10, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore anti synphilin 1 antibody
    Kalirin-7 interacts with <t>synphilin-1</t> in vitro and in vivo . (A) Mapping of the interacting domain in the kalirin-7 protein. FLAG-kalirin-7 constructs as shown in the diagram were co-transfected with V5-synphilin-1 in HEK293 cells. 24 h after transfection, cells were subjected to immunoprecipitation with anti-FLAG agarose beads and subsequently kalirin-7 and synphilin-1 immunoreactivities were monitored applying anti-FLAG- or anti-V5 antibodies, respectively. IP indicates antibodies used for pulling down target proteins. IB indicates antibodies used for detection in western blot. The figure shows that kalirin-7 co-immunopreciptates with synphilin-1 and that spectrin repeats III and IV of the kalirin-7 protein are crucial for the interaction. Quantification of kalirin-7 fragment expression is shown in Figure S3 . (B) Mapping of the binding region in synphilin-1. The indicated V5-synphilin-1 constructs were co-transfected with FLAG-kalirin-7. Synphilin-1 fragments were precipitated with anti-V5 antibodies. The precipitates were then probed with anti-FLAG antibodies to detect co-precipitated kalirin-7. The deletion mapping revealed that amino acids 1–348 of the synphilin-1 protein are crucial for the binding of kalirin-7. The asterisks indicate specific input signals of synphilin-1 fragments. For quantification of synphilin-1 fragment expression please refer to Figure S3 . (C) Endogenous synphilin-1 interacts with kalirin-7. Synphilin-1 was precipitated from whole-brain tissues (500 µg) of a wild type mouse with an anti-synphilin-1 antibody (Sigma). The precipitates were probed with a kalirin-7-specific antibody (KALRN from Abcam). Cell lysate of HEK293 cells overexpressed with FLAG-kalirin-7 and V5-synphilin-1 served as positive control. As a negative control brain lysate was subjected to immunoprecipitation without antibody. (D) Overlapping localization of kalirin-7 and synphilin-1 in cell culture. HEK293 cells were transiently transfected with both constructs for 6 h and stained with anti-FLAG and anti-V5 antibodies. The counterstaining was done with YoPro dye. The confocal sections demonstrate that both proteins display a punctate staining in the cytoplasm. Sph1, synphilin-1; Kal7, kalirin-7. Scale bar , 10 µm.
    Anti Synphilin 1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Reciprocal regulation of ephrin-A1 and EphA2 expression in corneal epithelial cell cultures. (A) Immunostaining of EphA2 (green), ephrin-A1 (red), and E-cadherin (magenta) in mono-cultures of hTCEPi cells transduced with an empty control or an ephrin-A1 cDNA construct. Representative images from n = 3. (B) Immunoblotting for total EphA2, ephrin-A1, or E-cadherin in hTCEPi cells overexpressing ephrin-A1 (EFNA1). GAPDH was used as a protein loading control. Representative blots from n = 4. (C) Immunostaining of EphA2 (green), ephrin-A1 (red), and E-cadherin (magenta) in hTCEPi cells knocked down for ephrin-A1 (siEphrin-A1), EphA2 (siEphA2), or both proteins (siEphA2+siEphrin-A1). Scale bar denotes 100 μm. Representative images from n = 3. (D) Immunoblotting for total EphA2, ephrin-A1, or E-cadherin in hTCEPi cells with siRNA targeted knockdown of ephrin-A1, EphA2, or double knockdown. GAPDH was used as a protein loading control. Representative blots from n = 3.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

    doi: 10.1167/iovs.17-22941

    Figure Lengend Snippet: Reciprocal regulation of ephrin-A1 and EphA2 expression in corneal epithelial cell cultures. (A) Immunostaining of EphA2 (green), ephrin-A1 (red), and E-cadherin (magenta) in mono-cultures of hTCEPi cells transduced with an empty control or an ephrin-A1 cDNA construct. Representative images from n = 3. (B) Immunoblotting for total EphA2, ephrin-A1, or E-cadherin in hTCEPi cells overexpressing ephrin-A1 (EFNA1). GAPDH was used as a protein loading control. Representative blots from n = 4. (C) Immunostaining of EphA2 (green), ephrin-A1 (red), and E-cadherin (magenta) in hTCEPi cells knocked down for ephrin-A1 (siEphrin-A1), EphA2 (siEphA2), or both proteins (siEphA2+siEphrin-A1). Scale bar denotes 100 μm. Representative images from n = 3. (D) Immunoblotting for total EphA2, ephrin-A1, or E-cadherin in hTCEPi cells with siRNA targeted knockdown of ephrin-A1, EphA2, or double knockdown. GAPDH was used as a protein loading control. Representative blots from n = 3.

    Article Snippet: Protein lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, pH 7.0, 150 mM NaCl, 1% Nonident-P 40, 0.1% SDS, 1% sodium deoxycholate, 5 mM EDTA, containing 1× protease inhibitor cocktail and 1× phosphatase inhibitor cocktail; Roche, Indianapolis, IN, USA) from hTCEpi cells and uninjured control or injured corneas, and subjected to Western blot analysis as previously described., Briefly, 5 to 25 μg protein lysate was separated by SDS-PAGE and probed with the following primary antibodies: mouse monoclonal antibody against EphA2 (D7; Millipore); rabbit polyclonal antibodies against ephrin-A1 (V18; Santa Cruz Biotechnologies), pSer897-EphA2 (Cell Signaling Technology), E-cadherin (HECD1; Abcam, Cambridge, MA, USA), and ERK1/2 (Cell Signaling Technologies) and GAPDH (Santa Cruz Biotechnologies) as loading controls.

    Techniques: Expressing, Immunostaining, Transduction, Construct

    Cell–cell border localization of E-cadherin is reduced at EphA2/Ephrin-A1 boundaries. (A) Immunofluorescent staining of EphA2 (green), ephrin-A1 (red), and E-cadherin (magenta) in cells present at the boundary of Control:Control- or Control:Ephrin-A1–expressing cell cocultures 48 hours after removal of the silicone barrier. A magnified view of the boundary is shown below in control cells confronting ephrin-A1–expressing cells. Dotted lines indicate the boundary between the two different cell populations 48 hours after initiation of confrontation. n = 4. Scale bar denotes 80 μm. (B) Control or ephrin-A1–expressing cells were transduced to express mCherry (Control-mCherry or EFNA1-mCherry, respectively) to differentiate these cell populations from control cells transfected with siControl (siCTRL) or siEphA2. Immunostaining of E-cadherin (magenta) was performed in cocultures 48 hours after initiation of confrontation. Dotted lines indicate the boundary between the two different cell populations at 48 hours. n = 3.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

    doi: 10.1167/iovs.17-22941

    Figure Lengend Snippet: Cell–cell border localization of E-cadherin is reduced at EphA2/Ephrin-A1 boundaries. (A) Immunofluorescent staining of EphA2 (green), ephrin-A1 (red), and E-cadherin (magenta) in cells present at the boundary of Control:Control- or Control:Ephrin-A1–expressing cell cocultures 48 hours after removal of the silicone barrier. A magnified view of the boundary is shown below in control cells confronting ephrin-A1–expressing cells. Dotted lines indicate the boundary between the two different cell populations 48 hours after initiation of confrontation. n = 4. Scale bar denotes 80 μm. (B) Control or ephrin-A1–expressing cells were transduced to express mCherry (Control-mCherry or EFNA1-mCherry, respectively) to differentiate these cell populations from control cells transfected with siControl (siCTRL) or siEphA2. Immunostaining of E-cadherin (magenta) was performed in cocultures 48 hours after initiation of confrontation. Dotted lines indicate the boundary between the two different cell populations at 48 hours. n = 3.

    Article Snippet: Protein lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, pH 7.0, 150 mM NaCl, 1% Nonident-P 40, 0.1% SDS, 1% sodium deoxycholate, 5 mM EDTA, containing 1× protease inhibitor cocktail and 1× phosphatase inhibitor cocktail; Roche, Indianapolis, IN, USA) from hTCEpi cells and uninjured control or injured corneas, and subjected to Western blot analysis as previously described., Briefly, 5 to 25 μg protein lysate was separated by SDS-PAGE and probed with the following primary antibodies: mouse monoclonal antibody against EphA2 (D7; Millipore); rabbit polyclonal antibodies against ephrin-A1 (V18; Santa Cruz Biotechnologies), pSer897-EphA2 (Cell Signaling Technology), E-cadherin (HECD1; Abcam, Cambridge, MA, USA), and ERK1/2 (Cell Signaling Technologies) and GAPDH (Santa Cruz Biotechnologies) as loading controls.

    Techniques: Staining, Expressing, Transfection, Immunostaining

    Ephrin-A1–induced boundary formation requires EphA2. (A) Control (Control, red) or (B) ephrin-A1–expressing (EFNA1, red) cells are shown at 48 hours after initiation of confrontation with cells transfected with siControl (siCTRL, green), siEphA2 (green), siEphrin-A1 (siEFNA1, green), or double siRNA (siEphA2+siEFNA1, green) oligonucleotides. Solid white lines mark the midline where the silicone divider was present at the time of removal. White dotted lines indicate the boundary between two cell populations 48 hours after initiation of confrontation. (C) Quantification of confrontation response in A and B as measured by % deviation from migration front of red-labeled cells (Control, red or Ephrin-A1, red). *P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

    doi: 10.1167/iovs.17-22941

    Figure Lengend Snippet: Ephrin-A1–induced boundary formation requires EphA2. (A) Control (Control, red) or (B) ephrin-A1–expressing (EFNA1, red) cells are shown at 48 hours after initiation of confrontation with cells transfected with siControl (siCTRL, green), siEphA2 (green), siEphrin-A1 (siEFNA1, green), or double siRNA (siEphA2+siEFNA1, green) oligonucleotides. Solid white lines mark the midline where the silicone divider was present at the time of removal. White dotted lines indicate the boundary between two cell populations 48 hours after initiation of confrontation. (C) Quantification of confrontation response in A and B as measured by % deviation from migration front of red-labeled cells (Control, red or Ephrin-A1, red). *P

    Article Snippet: Protein lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, pH 7.0, 150 mM NaCl, 1% Nonident-P 40, 0.1% SDS, 1% sodium deoxycholate, 5 mM EDTA, containing 1× protease inhibitor cocktail and 1× phosphatase inhibitor cocktail; Roche, Indianapolis, IN, USA) from hTCEpi cells and uninjured control or injured corneas, and subjected to Western blot analysis as previously described., Briefly, 5 to 25 μg protein lysate was separated by SDS-PAGE and probed with the following primary antibodies: mouse monoclonal antibody against EphA2 (D7; Millipore); rabbit polyclonal antibodies against ephrin-A1 (V18; Santa Cruz Biotechnologies), pSer897-EphA2 (Cell Signaling Technology), E-cadherin (HECD1; Abcam, Cambridge, MA, USA), and ERK1/2 (Cell Signaling Technologies) and GAPDH (Santa Cruz Biotechnologies) as loading controls.

    Techniques: Expressing, Transfection, Migration, Labeling

    Reciprocal regulation of ephrin-A1 and EphA2 expression in human and mouse cornea. Frozen corneal tissue sections from human cadavers (A) and wild-type Balb/C mice (B) were immunostained with antibodies against EphA2 or ephrin-A1 (red, bottom). DAPI (blue) was used to highlight nuclei. (A) Arrowheads indicate the limbus–cornea junction where the limbus ends and the cornea begins. (B) Mouse eyelids are marked as a reference point for limbal tissue orientation. Arrows show concentrated ephrin-A1 staining and paucity of EphA2 staining in the limbus. White dotted lines demarcate the basement membrane region. CC, central cornea; L, limbus. n = 3. Scale bar denotes 100 μm.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

    doi: 10.1167/iovs.17-22941

    Figure Lengend Snippet: Reciprocal regulation of ephrin-A1 and EphA2 expression in human and mouse cornea. Frozen corneal tissue sections from human cadavers (A) and wild-type Balb/C mice (B) were immunostained with antibodies against EphA2 or ephrin-A1 (red, bottom). DAPI (blue) was used to highlight nuclei. (A) Arrowheads indicate the limbus–cornea junction where the limbus ends and the cornea begins. (B) Mouse eyelids are marked as a reference point for limbal tissue orientation. Arrows show concentrated ephrin-A1 staining and paucity of EphA2 staining in the limbus. White dotted lines demarcate the basement membrane region. CC, central cornea; L, limbus. n = 3. Scale bar denotes 100 μm.

    Article Snippet: Protein lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, pH 7.0, 150 mM NaCl, 1% Nonident-P 40, 0.1% SDS, 1% sodium deoxycholate, 5 mM EDTA, containing 1× protease inhibitor cocktail and 1× phosphatase inhibitor cocktail; Roche, Indianapolis, IN, USA) from hTCEpi cells and uninjured control or injured corneas, and subjected to Western blot analysis as previously described., Briefly, 5 to 25 μg protein lysate was separated by SDS-PAGE and probed with the following primary antibodies: mouse monoclonal antibody against EphA2 (D7; Millipore); rabbit polyclonal antibodies against ephrin-A1 (V18; Santa Cruz Biotechnologies), pSer897-EphA2 (Cell Signaling Technology), E-cadherin (HECD1; Abcam, Cambridge, MA, USA), and ERK1/2 (Cell Signaling Technologies) and GAPDH (Santa Cruz Biotechnologies) as loading controls.

    Techniques: Expressing, Mouse Assay, Staining

    ADAM10 mediates Ephrin-A1/EphA2 boundary organization via EGFR signaling. (A) E-cadherin (E-cad; top) and ADAM10 (bottom) immunofluorescence staining in human anterior segmental epithelium. Scale bar denotes 100 μm. (B) E-cadherin staining of control cells (Control, green) confronted by “like” control cells (Control, red) or ephrin-A1–expressing cells (EFNA1, red) confronted by “unlike” control cells (Control, green; bottom) in the presence of general MMP inhibitor, TAPI, or a specific ADAM10 inhibitor, GI254023X (GIX). Red dotted lines indicate the boundary between the two cell populations 48 hours after initiation of confrontation. Scale bar denotes 80 μm. (C) Quantification of confrontation experiments at 48 hours in cocultures treated with DMSO, GIX, LY294002 (LY), Y-27632 (Y), or U0126 (U). * P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

    doi: 10.1167/iovs.17-22941

    Figure Lengend Snippet: ADAM10 mediates Ephrin-A1/EphA2 boundary organization via EGFR signaling. (A) E-cadherin (E-cad; top) and ADAM10 (bottom) immunofluorescence staining in human anterior segmental epithelium. Scale bar denotes 100 μm. (B) E-cadherin staining of control cells (Control, green) confronted by “like” control cells (Control, red) or ephrin-A1–expressing cells (EFNA1, red) confronted by “unlike” control cells (Control, green; bottom) in the presence of general MMP inhibitor, TAPI, or a specific ADAM10 inhibitor, GI254023X (GIX). Red dotted lines indicate the boundary between the two cell populations 48 hours after initiation of confrontation. Scale bar denotes 80 μm. (C) Quantification of confrontation experiments at 48 hours in cocultures treated with DMSO, GIX, LY294002 (LY), Y-27632 (Y), or U0126 (U). * P

    Article Snippet: Protein lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, pH 7.0, 150 mM NaCl, 1% Nonident-P 40, 0.1% SDS, 1% sodium deoxycholate, 5 mM EDTA, containing 1× protease inhibitor cocktail and 1× phosphatase inhibitor cocktail; Roche, Indianapolis, IN, USA) from hTCEpi cells and uninjured control or injured corneas, and subjected to Western blot analysis as previously described., Briefly, 5 to 25 μg protein lysate was separated by SDS-PAGE and probed with the following primary antibodies: mouse monoclonal antibody against EphA2 (D7; Millipore); rabbit polyclonal antibodies against ephrin-A1 (V18; Santa Cruz Biotechnologies), pSer897-EphA2 (Cell Signaling Technology), E-cadherin (HECD1; Abcam, Cambridge, MA, USA), and ERK1/2 (Cell Signaling Technologies) and GAPDH (Santa Cruz Biotechnologies) as loading controls.

    Techniques: Immunofluorescence, Staining, Expressing

    EphA2/Ephrin-A1 signaling complexes in a heterotypic cell confrontation coculture model. (A) hTCEpi cells were differentially labeled with fluorescent cell trackers (red and green) and seeded into discrete culture compartments using a silicone chamber confrontation apparatus. After removal of the silicone divider, live cell imaging was used to monitor cell confrontation for 48 hours. Snapshots of 0, 24, and 48 hours are shown. (B) EphA2-expressing control cells (Control, green) confronting “like” control cells (Control, red) are presented on the left, while control cells (Control, green) confronting “unlike” ephrin-A1–overexpressing cells (EFNA1, red) are presented on the right. After removal of the silicone divider, time-lapse imaging was used to examine the formation and organization of the epithelial boundary between these two cell populations. White solid lines mark the midline where the silicone divider was present prior to removal. Dotted lines indicate the boundary between the two cell populations after initiation of confrontation. Snapshots of 0, 6, 12, 24, and 48 hours are shown. Scale bar denotes 1 mm. (C, D) Line graphs showing the migrating front of control cells (Control, green) confronting control (Control, red) (C) or ephrin-A1 (EFNA1, red) (D) overexpressing cells normalized with respect to the midline. Negative values on the y-axis represent the reversal of migration initiated by ephrin-A1. n = 3 (D). (E) Higher-magnification images from early time points (2, 3, 4, 6, 8, and 12 hours) of ephrin-A1–overexpressing cells (EFNA1, red) confronting control cells (CTRL, green). Arrowhead points to the initial confrontation area at 4 hours. Scale bar denotes 200 μm.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

    doi: 10.1167/iovs.17-22941

    Figure Lengend Snippet: EphA2/Ephrin-A1 signaling complexes in a heterotypic cell confrontation coculture model. (A) hTCEpi cells were differentially labeled with fluorescent cell trackers (red and green) and seeded into discrete culture compartments using a silicone chamber confrontation apparatus. After removal of the silicone divider, live cell imaging was used to monitor cell confrontation for 48 hours. Snapshots of 0, 24, and 48 hours are shown. (B) EphA2-expressing control cells (Control, green) confronting “like” control cells (Control, red) are presented on the left, while control cells (Control, green) confronting “unlike” ephrin-A1–overexpressing cells (EFNA1, red) are presented on the right. After removal of the silicone divider, time-lapse imaging was used to examine the formation and organization of the epithelial boundary between these two cell populations. White solid lines mark the midline where the silicone divider was present prior to removal. Dotted lines indicate the boundary between the two cell populations after initiation of confrontation. Snapshots of 0, 6, 12, 24, and 48 hours are shown. Scale bar denotes 1 mm. (C, D) Line graphs showing the migrating front of control cells (Control, green) confronting control (Control, red) (C) or ephrin-A1 (EFNA1, red) (D) overexpressing cells normalized with respect to the midline. Negative values on the y-axis represent the reversal of migration initiated by ephrin-A1. n = 3 (D). (E) Higher-magnification images from early time points (2, 3, 4, 6, 8, and 12 hours) of ephrin-A1–overexpressing cells (EFNA1, red) confronting control cells (CTRL, green). Arrowhead points to the initial confrontation area at 4 hours. Scale bar denotes 200 μm.

    Article Snippet: Protein lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, pH 7.0, 150 mM NaCl, 1% Nonident-P 40, 0.1% SDS, 1% sodium deoxycholate, 5 mM EDTA, containing 1× protease inhibitor cocktail and 1× phosphatase inhibitor cocktail; Roche, Indianapolis, IN, USA) from hTCEpi cells and uninjured control or injured corneas, and subjected to Western blot analysis as previously described., Briefly, 5 to 25 μg protein lysate was separated by SDS-PAGE and probed with the following primary antibodies: mouse monoclonal antibody against EphA2 (D7; Millipore); rabbit polyclonal antibodies against ephrin-A1 (V18; Santa Cruz Biotechnologies), pSer897-EphA2 (Cell Signaling Technology), E-cadherin (HECD1; Abcam, Cambridge, MA, USA), and ERK1/2 (Cell Signaling Technologies) and GAPDH (Santa Cruz Biotechnologies) as loading controls.

    Techniques: Labeling, Live Cell Imaging, Expressing, Imaging, Migration

    Ephrin-A1 is redistributed into the cornea of injured mouse eyes. Whole mounts of mouse anterior segmental epithelium in uninjured (Control) eyes and following central corneal wounding (24 hours after injury). Immunostaining was performed for (A) EphA2 (red) and (B) Ephrin-A1 (red). Scale bar denotes 80 μm. W, wound opening. Arrowheads show clusters of ephrin-A1–expressing cells in the cornea. Arrow represents EphA2-enriched areas near the wound edge. White dotted line marks the wound edge. n = 3. (C) Whole mounts of control (upper) and injured (lower) mouse corneas that were dual stained for EphA2 (red) and ephrin-A1 (green). Confocal stitched images show the entire cornea. L, limbus; PC, peripheral cornea; CC, central cornea; W, wound opening. White dotted line marks the wound edge. (D) Immunoblotting of EphA2, pS897-EphA2, or ephrin-A1 in samples isolated from wounded corneas. ERK1/2 was used as a loading control. (E) Densitometry results of Western blots represented in D are shown as fold changes over levels present in uninjured, control corneas. *P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

    doi: 10.1167/iovs.17-22941

    Figure Lengend Snippet: Ephrin-A1 is redistributed into the cornea of injured mouse eyes. Whole mounts of mouse anterior segmental epithelium in uninjured (Control) eyes and following central corneal wounding (24 hours after injury). Immunostaining was performed for (A) EphA2 (red) and (B) Ephrin-A1 (red). Scale bar denotes 80 μm. W, wound opening. Arrowheads show clusters of ephrin-A1–expressing cells in the cornea. Arrow represents EphA2-enriched areas near the wound edge. White dotted line marks the wound edge. n = 3. (C) Whole mounts of control (upper) and injured (lower) mouse corneas that were dual stained for EphA2 (red) and ephrin-A1 (green). Confocal stitched images show the entire cornea. L, limbus; PC, peripheral cornea; CC, central cornea; W, wound opening. White dotted line marks the wound edge. (D) Immunoblotting of EphA2, pS897-EphA2, or ephrin-A1 in samples isolated from wounded corneas. ERK1/2 was used as a loading control. (E) Densitometry results of Western blots represented in D are shown as fold changes over levels present in uninjured, control corneas. *P

    Article Snippet: Protein lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, pH 7.0, 150 mM NaCl, 1% Nonident-P 40, 0.1% SDS, 1% sodium deoxycholate, 5 mM EDTA, containing 1× protease inhibitor cocktail and 1× phosphatase inhibitor cocktail; Roche, Indianapolis, IN, USA) from hTCEpi cells and uninjured control or injured corneas, and subjected to Western blot analysis as previously described., Briefly, 5 to 25 μg protein lysate was separated by SDS-PAGE and probed with the following primary antibodies: mouse monoclonal antibody against EphA2 (D7; Millipore); rabbit polyclonal antibodies against ephrin-A1 (V18; Santa Cruz Biotechnologies), pSer897-EphA2 (Cell Signaling Technology), E-cadherin (HECD1; Abcam, Cambridge, MA, USA), and ERK1/2 (Cell Signaling Technologies) and GAPDH (Santa Cruz Biotechnologies) as loading controls.

    Techniques: Immunostaining, Expressing, Staining, Isolation, Western Blot

    The NGFR High phenotype can be suppressed by JQ 1 in BRAF V 600E melanoma xenografts Immunofluorescence analysis of A375 melanoma xenograft tumors co‐stained for Ki‐67 and NGFR proteins. Selected images from a whole tumor section as well as NGFR High /Ki‐67 Low and NGFR Low /Ki‐67 High regions of a vehicle‐treated tumor are shown to highlight the spatial and cell‐to‐cell heterogeneity in Ki‐67 and NGFR protein expression. Percentage of Ki‐67 High and NGFR High cells in tumors treated for 5 days with dabrafenib (25 mg/kg) only, dabrafenib (25 mg/kg) in combination with JQ1 (50 mg/kg), or vehicle. Number of tumors (mice) analyzed per condition is shown. Solid horizontal lines represent the mean of measurements. Up to 50,000 individual cells per tumor were analyzed for NGFR and Ki‐67 intensities. Statistical significance was determined using two‐tailed two‐sample t ‐test.

    Journal: Molecular Systems Biology

    Article Title: Adaptive resistance of melanoma cells to RAF inhibition via reversible induction of a slowly dividing de‐differentiated state

    doi: 10.15252/msb.20166796

    Figure Lengend Snippet: The NGFR High phenotype can be suppressed by JQ 1 in BRAF V 600E melanoma xenografts Immunofluorescence analysis of A375 melanoma xenograft tumors co‐stained for Ki‐67 and NGFR proteins. Selected images from a whole tumor section as well as NGFR High /Ki‐67 Low and NGFR Low /Ki‐67 High regions of a vehicle‐treated tumor are shown to highlight the spatial and cell‐to‐cell heterogeneity in Ki‐67 and NGFR protein expression. Percentage of Ki‐67 High and NGFR High cells in tumors treated for 5 days with dabrafenib (25 mg/kg) only, dabrafenib (25 mg/kg) in combination with JQ1 (50 mg/kg), or vehicle. Number of tumors (mice) analyzed per condition is shown. Solid horizontal lines represent the mean of measurements. Up to 50,000 individual cells per tumor were analyzed for NGFR and Ki‐67 intensities. Statistical significance was determined using two‐tailed two‐sample t ‐test.

    Article Snippet: Sections were incubated for 30 min with primary antibodies including Ki‐67 rabbit monoclonal antibody (clone SP6, Cat# VP‐RM04) from Vector Laboratories, p75NTR/NGFR rabbit polyclonal antibody (Cat# 119‐11668) from RayBiotech, and MITF mouse monoclonal antibody (clone D5, Cat# MA5‐14154) from Thermo Scientific, and were then completed with the Leica Refine detection kit (secondary antibody, the DAB chromogen, and the hematoxylin counterstain).

    Techniques: Immunofluorescence, Staining, Expressing, Mouse Assay, Two Tailed Test

    Concurrent inhibition of RAF / MEK signaling and the c‐Jun/ FAK /Src cascade blocks the NGFR High state and increases cell killing NGFR levels as measured by immunofluorescence (left panel) and relative cell viability (right panel) in COLO858 cells following treatment in duplicate with indicated doses of vemurafenib in the presence of siRNAs targeting JUN , PTK2, and NGFR for 72 h. Viability data for each siRNA condition at each dose of vemurafenib were normalized to cells treated with the same dose and the non‐targeting siRNA. NGFR protein levels measured by immunofluorescence in duplicate in COLO858 cells treated for 48 h with indicated doses of vemurafenib, in combination with DMSO, MEK inhibitor trametinib (0.6 μM), FAK inhibitors defactinib (3 μM) and PF562271 (3 μM), JNK inhibitor JNK‐IN‐8 (3 μM), or Src inhibitors dasatinib (3 μM) and saracatinib (3 μM). Pairwise comparison between drug combination‐induced changes in NGFR and Ki‐67 in COLO858 cells treated for 48 h with vemurafenib at 0.32 and 1 μM in combination with DMSO or two doses of trametinib (0.2, 0.6 μM), defactinib (1, 3 μM), PF562271 (1, 3 μM), dasatinib (1, 3 μM), saracatinib (1, 3 μM), and JNK‐IN‐8 (1, 3 μM). NGFR and Ki‐67 levels were measured by immunofluorescence. For each signal, data were averaged across two replicates, two doses of vemurafenib, and two doses of the second drug, log‐transformed, and z ‐score‐scaled across seven different drug combinations. Relative viability of COLO858 cells treated for 72 h with vemurafenib or vemurafenib plus trametinib (10:1 dose ratio) in combination with DMSO, JNK‐IN‐8, dasatinib, saracatinib, and defactinib at indicated doses. Viability data were measured in three replicates and normalized to DMSO‐treated controls. Data information: Data in (A, B, D) are presented as mean ± SD. Statistical significance was determined by two‐way ANOVA.

    Journal: Molecular Systems Biology

    Article Title: Adaptive resistance of melanoma cells to RAF inhibition via reversible induction of a slowly dividing de‐differentiated state

    doi: 10.15252/msb.20166796

    Figure Lengend Snippet: Concurrent inhibition of RAF / MEK signaling and the c‐Jun/ FAK /Src cascade blocks the NGFR High state and increases cell killing NGFR levels as measured by immunofluorescence (left panel) and relative cell viability (right panel) in COLO858 cells following treatment in duplicate with indicated doses of vemurafenib in the presence of siRNAs targeting JUN , PTK2, and NGFR for 72 h. Viability data for each siRNA condition at each dose of vemurafenib were normalized to cells treated with the same dose and the non‐targeting siRNA. NGFR protein levels measured by immunofluorescence in duplicate in COLO858 cells treated for 48 h with indicated doses of vemurafenib, in combination with DMSO, MEK inhibitor trametinib (0.6 μM), FAK inhibitors defactinib (3 μM) and PF562271 (3 μM), JNK inhibitor JNK‐IN‐8 (3 μM), or Src inhibitors dasatinib (3 μM) and saracatinib (3 μM). Pairwise comparison between drug combination‐induced changes in NGFR and Ki‐67 in COLO858 cells treated for 48 h with vemurafenib at 0.32 and 1 μM in combination with DMSO or two doses of trametinib (0.2, 0.6 μM), defactinib (1, 3 μM), PF562271 (1, 3 μM), dasatinib (1, 3 μM), saracatinib (1, 3 μM), and JNK‐IN‐8 (1, 3 μM). NGFR and Ki‐67 levels were measured by immunofluorescence. For each signal, data were averaged across two replicates, two doses of vemurafenib, and two doses of the second drug, log‐transformed, and z ‐score‐scaled across seven different drug combinations. Relative viability of COLO858 cells treated for 72 h with vemurafenib or vemurafenib plus trametinib (10:1 dose ratio) in combination with DMSO, JNK‐IN‐8, dasatinib, saracatinib, and defactinib at indicated doses. Viability data were measured in three replicates and normalized to DMSO‐treated controls. Data information: Data in (A, B, D) are presented as mean ± SD. Statistical significance was determined by two‐way ANOVA.

    Article Snippet: Sections were incubated for 30 min with primary antibodies including Ki‐67 rabbit monoclonal antibody (clone SP6, Cat# VP‐RM04) from Vector Laboratories, p75NTR/NGFR rabbit polyclonal antibody (Cat# 119‐11668) from RayBiotech, and MITF mouse monoclonal antibody (clone D5, Cat# MA5‐14154) from Thermo Scientific, and were then completed with the Leica Refine detection kit (secondary antibody, the DAB chromogen, and the hematoxylin counterstain).

    Techniques: Inhibition, Immunofluorescence, Transformation Assay

    The NGFR High drug‐resistant state is dependent on AP1 and focal adhesion signaling, but not NGF signaling A list of transcription factor candidates predicted to regulate differentially expressed receptors and secreted factors between vemurafenib‐treated COLO858 and MMACSF cells. Western blotting for NGFR‐inducible COLO858 cells, NGFR High A375 and WM115 cells, and NGFR Low MMACSF and MZ7MEL cells, treated for 48 h with 0.2 or 1 μM vemurafenib or DMSO. The effect of NGF at indicated concentrations on viability of COLO858 and MMACSF cells treated in duplicate with vemurafenib at indicated doses for 48 h. Data are presented as mean ± SD. Statistical significance was determined by two‐way ANOVA.

    Journal: Molecular Systems Biology

    Article Title: Adaptive resistance of melanoma cells to RAF inhibition via reversible induction of a slowly dividing de‐differentiated state

    doi: 10.15252/msb.20166796

    Figure Lengend Snippet: The NGFR High drug‐resistant state is dependent on AP1 and focal adhesion signaling, but not NGF signaling A list of transcription factor candidates predicted to regulate differentially expressed receptors and secreted factors between vemurafenib‐treated COLO858 and MMACSF cells. Western blotting for NGFR‐inducible COLO858 cells, NGFR High A375 and WM115 cells, and NGFR Low MMACSF and MZ7MEL cells, treated for 48 h with 0.2 or 1 μM vemurafenib or DMSO. The effect of NGF at indicated concentrations on viability of COLO858 and MMACSF cells treated in duplicate with vemurafenib at indicated doses for 48 h. Data are presented as mean ± SD. Statistical significance was determined by two‐way ANOVA.

    Article Snippet: Sections were incubated for 30 min with primary antibodies including Ki‐67 rabbit monoclonal antibody (clone SP6, Cat# VP‐RM04) from Vector Laboratories, p75NTR/NGFR rabbit polyclonal antibody (Cat# 119‐11668) from RayBiotech, and MITF mouse monoclonal antibody (clone D5, Cat# MA5‐14154) from Thermo Scientific, and were then completed with the Leica Refine detection kit (secondary antibody, the DAB chromogen, and the hematoxylin counterstain).

    Techniques: Western Blot

    Drug resistance is associated with de‐differentiation of cells to a slowly cycling NGFR High phenotype Differentially up‐regulated genes in COLO858 relative to MMACSF cells treated with 0.2 μM vemurafenib for 24 and 48 h (log 2 (ratio) ≥ 1). Selected genes involved in neurogenesis, neural differentiation and myelination (red), cell adhesion, ECM remodeling and epithelial–mesenchymal transition (brown), and cell cycle regulation (blue) are highlighted. Top Gene Ontology (GO) biological processes differentially regulated between COLO858 and MMACSF cells. NGFR protein levels measured in duplicate by immunofluorescence in COLO858 and MMACSF cells treated with indicated doses of vemurafenib for 48 or 72 h. Data are presented as mean ± SD. Covariate single‐cell analysis of Ki‐67 versus NGFR in COLO858 cells 24–72 h after exposure to 1 μM vemurafenib or DMSO.

    Journal: Molecular Systems Biology

    Article Title: Adaptive resistance of melanoma cells to RAF inhibition via reversible induction of a slowly dividing de‐differentiated state

    doi: 10.15252/msb.20166796

    Figure Lengend Snippet: Drug resistance is associated with de‐differentiation of cells to a slowly cycling NGFR High phenotype Differentially up‐regulated genes in COLO858 relative to MMACSF cells treated with 0.2 μM vemurafenib for 24 and 48 h (log 2 (ratio) ≥ 1). Selected genes involved in neurogenesis, neural differentiation and myelination (red), cell adhesion, ECM remodeling and epithelial–mesenchymal transition (brown), and cell cycle regulation (blue) are highlighted. Top Gene Ontology (GO) biological processes differentially regulated between COLO858 and MMACSF cells. NGFR protein levels measured in duplicate by immunofluorescence in COLO858 and MMACSF cells treated with indicated doses of vemurafenib for 48 or 72 h. Data are presented as mean ± SD. Covariate single‐cell analysis of Ki‐67 versus NGFR in COLO858 cells 24–72 h after exposure to 1 μM vemurafenib or DMSO.

    Article Snippet: Sections were incubated for 30 min with primary antibodies including Ki‐67 rabbit monoclonal antibody (clone SP6, Cat# VP‐RM04) from Vector Laboratories, p75NTR/NGFR rabbit polyclonal antibody (Cat# 119‐11668) from RayBiotech, and MITF mouse monoclonal antibody (clone D5, Cat# MA5‐14154) from Thermo Scientific, and were then completed with the Leica Refine detection kit (secondary antibody, the DAB chromogen, and the hematoxylin counterstain).

    Techniques: Immunofluorescence, Single-cell Analysis

    The NGFR High state involves extracellular matrix ( ECM ) components, focal adhesion, and the AP 1 transcription factor c‐Jun Top differentially regulated genes encoding secreted proteins (A) and cell surface receptors (B) between COLO858 and MMACSF cells. Ranked GSEA plots of top KEGG pathways significantly correlated with NGFR expression in 25 BRAF V600E melanoma cell lines from the CCLE (top) and tumor biopsies of 128 BRAF V600E melanoma patients in TCGA (bottom). A list of transcription factor candidates predicted (by DAVID; see Materials and Methods ) to regulate differentially expressed genes between vemurafenib‐treated COLO858 and MMACSF cells (D), and the corresponding transcription factor gene expression levels in these cells (E). Quantified Western blot measurements (see Materials and Methods ) for thrombospondin‐1 (THBS1; TSP‐1), integrin β1, and p‐FAK Y397 in COLO858 and MMACSF cells treated for 48 h with indicated doses of vemurafenib. Data are first normalized to HSP90α/β levels in each cell line at each treatment condition and then to DMSO‐treated COLO858 cells. c‐Jun and p‐c‐Jun S73 changes as measured in duplicate by immunofluorescence in COLO858 and MMACSF cells treated for 48 h with indicated doses of vemurafenib. Data are normalized to DMSO‐treated COLO858 cells. Data information: Data in (F, G) are presented as mean ± SD.

    Journal: Molecular Systems Biology

    Article Title: Adaptive resistance of melanoma cells to RAF inhibition via reversible induction of a slowly dividing de‐differentiated state

    doi: 10.15252/msb.20166796

    Figure Lengend Snippet: The NGFR High state involves extracellular matrix ( ECM ) components, focal adhesion, and the AP 1 transcription factor c‐Jun Top differentially regulated genes encoding secreted proteins (A) and cell surface receptors (B) between COLO858 and MMACSF cells. Ranked GSEA plots of top KEGG pathways significantly correlated with NGFR expression in 25 BRAF V600E melanoma cell lines from the CCLE (top) and tumor biopsies of 128 BRAF V600E melanoma patients in TCGA (bottom). A list of transcription factor candidates predicted (by DAVID; see Materials and Methods ) to regulate differentially expressed genes between vemurafenib‐treated COLO858 and MMACSF cells (D), and the corresponding transcription factor gene expression levels in these cells (E). Quantified Western blot measurements (see Materials and Methods ) for thrombospondin‐1 (THBS1; TSP‐1), integrin β1, and p‐FAK Y397 in COLO858 and MMACSF cells treated for 48 h with indicated doses of vemurafenib. Data are first normalized to HSP90α/β levels in each cell line at each treatment condition and then to DMSO‐treated COLO858 cells. c‐Jun and p‐c‐Jun S73 changes as measured in duplicate by immunofluorescence in COLO858 and MMACSF cells treated for 48 h with indicated doses of vemurafenib. Data are normalized to DMSO‐treated COLO858 cells. Data information: Data in (F, G) are presented as mean ± SD.

    Article Snippet: Sections were incubated for 30 min with primary antibodies including Ki‐67 rabbit monoclonal antibody (clone SP6, Cat# VP‐RM04) from Vector Laboratories, p75NTR/NGFR rabbit polyclonal antibody (Cat# 119‐11668) from RayBiotech, and MITF mouse monoclonal antibody (clone D5, Cat# MA5‐14154) from Thermo Scientific, and were then completed with the Leica Refine detection kit (secondary antibody, the DAB chromogen, and the hematoxylin counterstain).

    Techniques: Expressing, Western Blot, Immunofluorescence

    JNK , FAK , Src, and BET inhibitors overcome the NGFR High drug‐resistant state in additional BRAF V 600E/D melanoma lines NGFR protein levels measured in duplicate by immunofluorescence in seven BRAF V600E/D cell lines treated with vemurafenib at indicated doses for 48 h. Correlation between vemurafenib‐induced changes in c‐Jun and NGFR protein levels across nine BRAF V600E/D melanoma cell lines. Cells were treated with five doses of vemurafenib (0, 0.1, 0.32, 1, and 3.2 μM) for 48 h. c‐Jun and NGFR protein levels measured by immunofluorescence at each condition were averaged across two replicates and normalized to DMSO‐treated controls. The area under the dose–response curve (AUC) for the two measurements (c‐Jun and NGFR) was calculated, z ‐score‐scaled across nine cell lines, and their pairwise Pearson's correlation was reported. Relative viability of A375 and WM115 cells treated in 3 replicates for 72 h with vemurafenib or vemurafenib plus trametinib (10:1 dose ratio) in combination with indicated kinase inhibitors (C) or BET inhibitors (D). Pairwise comparison between NGFR and Ki‐67 levels in A375 and WM115 cells treated with vemurafenib in combination with indicated kinase inhibitors (E) or BET inhibitors (F). Drug doses, time points, and data normalization are similar to Figs 7 C and 8 C. Data information: Data in (A, C, D) are presented as mean ± SD.

    Journal: Molecular Systems Biology

    Article Title: Adaptive resistance of melanoma cells to RAF inhibition via reversible induction of a slowly dividing de‐differentiated state

    doi: 10.15252/msb.20166796

    Figure Lengend Snippet: JNK , FAK , Src, and BET inhibitors overcome the NGFR High drug‐resistant state in additional BRAF V 600E/D melanoma lines NGFR protein levels measured in duplicate by immunofluorescence in seven BRAF V600E/D cell lines treated with vemurafenib at indicated doses for 48 h. Correlation between vemurafenib‐induced changes in c‐Jun and NGFR protein levels across nine BRAF V600E/D melanoma cell lines. Cells were treated with five doses of vemurafenib (0, 0.1, 0.32, 1, and 3.2 μM) for 48 h. c‐Jun and NGFR protein levels measured by immunofluorescence at each condition were averaged across two replicates and normalized to DMSO‐treated controls. The area under the dose–response curve (AUC) for the two measurements (c‐Jun and NGFR) was calculated, z ‐score‐scaled across nine cell lines, and their pairwise Pearson's correlation was reported. Relative viability of A375 and WM115 cells treated in 3 replicates for 72 h with vemurafenib or vemurafenib plus trametinib (10:1 dose ratio) in combination with indicated kinase inhibitors (C) or BET inhibitors (D). Pairwise comparison between NGFR and Ki‐67 levels in A375 and WM115 cells treated with vemurafenib in combination with indicated kinase inhibitors (E) or BET inhibitors (F). Drug doses, time points, and data normalization are similar to Figs 7 C and 8 C. Data information: Data in (A, C, D) are presented as mean ± SD.

    Article Snippet: Sections were incubated for 30 min with primary antibodies including Ki‐67 rabbit monoclonal antibody (clone SP6, Cat# VP‐RM04) from Vector Laboratories, p75NTR/NGFR rabbit polyclonal antibody (Cat# 119‐11668) from RayBiotech, and MITF mouse monoclonal antibody (clone D5, Cat# MA5‐14154) from Thermo Scientific, and were then completed with the Leica Refine detection kit (secondary antibody, the DAB chromogen, and the hematoxylin counterstain).

    Techniques: Immunofluorescence

    BET inhibitors suppress the slowly cycling NGFR High state and effectively reduce the cancer cell population with time COLO858 cells were treated for 48 h in duplicate with vemurafenib (at 0.32 μM) in combination with DMSO or three doses (0.11, 0.53, and 2.67 μM) of each of 41 compounds in a chromatin‐targeting library. NGFR protein levels were measured by immunofluorescence, averaged across three doses of each compound, and z ‐scored. Relative viability of COLO858 cells treated for 72 h with vemurafenib or vemurafenib plus trametinib (10:1 dose ratio) in combination with DMSO, (+)‐JQ1, I‐BET, and I‐BET151 at indicated doses. Viability data were measured in three replicates and normalized to DMSO‐treated controls. Pairwise comparison between drug‐induced changes in NGFR and Ki‐67 in COLO858 cells treated with vemurafenib at 0.32, 1, and 3.2 μM in combination with DMSO or trametinib (0.2 μM), I‐BET (1 μM), I‐BET151 (1 μM), and (+)‐JQ1 (1 μM) for 48 h. Data for each drug combination were averaged across two replicates and three doses of vemurafenib, log‐transformed, and z ‐score‐scaled. c‐Jun protein levels measured by immunofluorescence in duplicate in COLO858 cells treated for 48 h with indicated doses of vemurafenib, in combination with DMSO, I‐BET (1 μM), (+)‐JQ1 (1 μM), and I‐BET151 (1 μM). Single‐cell analysis of division and death events following live‐cell imaging of COLO858 cells treated with 1 μM vemurafenib in combination with DMSO or (+)‐JQ1 (0.32 μM) for 84 h. Data are presented as described in Figure 1 . Time‐lapse analysis of COLO858 cells treated in three replicates for ˜1 week with different drug combinations at indicated doses. Data for DMSO‐treated cells are shown until day 3, the time at which cells reach ˜100% confluency. Data information: Data in (B, D, F) are presented as mean ± SD.

    Journal: Molecular Systems Biology

    Article Title: Adaptive resistance of melanoma cells to RAF inhibition via reversible induction of a slowly dividing de‐differentiated state

    doi: 10.15252/msb.20166796

    Figure Lengend Snippet: BET inhibitors suppress the slowly cycling NGFR High state and effectively reduce the cancer cell population with time COLO858 cells were treated for 48 h in duplicate with vemurafenib (at 0.32 μM) in combination with DMSO or three doses (0.11, 0.53, and 2.67 μM) of each of 41 compounds in a chromatin‐targeting library. NGFR protein levels were measured by immunofluorescence, averaged across three doses of each compound, and z ‐scored. Relative viability of COLO858 cells treated for 72 h with vemurafenib or vemurafenib plus trametinib (10:1 dose ratio) in combination with DMSO, (+)‐JQ1, I‐BET, and I‐BET151 at indicated doses. Viability data were measured in three replicates and normalized to DMSO‐treated controls. Pairwise comparison between drug‐induced changes in NGFR and Ki‐67 in COLO858 cells treated with vemurafenib at 0.32, 1, and 3.2 μM in combination with DMSO or trametinib (0.2 μM), I‐BET (1 μM), I‐BET151 (1 μM), and (+)‐JQ1 (1 μM) for 48 h. Data for each drug combination were averaged across two replicates and three doses of vemurafenib, log‐transformed, and z ‐score‐scaled. c‐Jun protein levels measured by immunofluorescence in duplicate in COLO858 cells treated for 48 h with indicated doses of vemurafenib, in combination with DMSO, I‐BET (1 μM), (+)‐JQ1 (1 μM), and I‐BET151 (1 μM). Single‐cell analysis of division and death events following live‐cell imaging of COLO858 cells treated with 1 μM vemurafenib in combination with DMSO or (+)‐JQ1 (0.32 μM) for 84 h. Data are presented as described in Figure 1 . Time‐lapse analysis of COLO858 cells treated in three replicates for ˜1 week with different drug combinations at indicated doses. Data for DMSO‐treated cells are shown until day 3, the time at which cells reach ˜100% confluency. Data information: Data in (B, D, F) are presented as mean ± SD.

    Article Snippet: Sections were incubated for 30 min with primary antibodies including Ki‐67 rabbit monoclonal antibody (clone SP6, Cat# VP‐RM04) from Vector Laboratories, p75NTR/NGFR rabbit polyclonal antibody (Cat# 119‐11668) from RayBiotech, and MITF mouse monoclonal antibody (clone D5, Cat# MA5‐14154) from Thermo Scientific, and were then completed with the Leica Refine detection kit (secondary antibody, the DAB chromogen, and the hematoxylin counterstain).

    Techniques: Immunofluorescence, Transformation Assay, Single-cell Analysis, Live Cell Imaging

    Vemurafenib‐induced de‐differentiation of cells and adaptive resistance are reversible upon drug removal Schematic outline of an experiment involving induction of the slowly cycling NGFR High state in COLO858 cells following 48‐h treatment with 0.32 μM vemurafenib, sorting cells to obtain NGFR Low and NGFR High subpopulations, recovering each cell subpopulation in fresh growth medium for 1–9 days, and re‐inducing recovered cells with vemurafenib. NGFR and Ki‐67 protein levels measured by immunofluorescence in cells grown for 9 days in fresh medium ( n = 4). Growth rate (GR) inhibition assay performed on FACS‐sorted NGFR High and NGFR Low pools of cells after 2 (C) or 9 (D) days of outgrowth in fresh medium. Measurements were performed in 4 (C) or 6 (D) replicates. Growth rate and doubling time measurements in 4 replicates in FACS‐sorted NGFR High and NGFR Low cells during 2–5 days of outgrowth in fresh medium. NGFR levels measured in duplicate by immunofluorescence in COLO858 cells recovered after 9 days of outgrowth in fresh media and subsequently re‐exposed for 48 h to four doses of vemurafenib. Data information: Data in (B–F) are presented as mean ± SD. Statistical significance was determined by two‐way ANOVA.

    Journal: Molecular Systems Biology

    Article Title: Adaptive resistance of melanoma cells to RAF inhibition via reversible induction of a slowly dividing de‐differentiated state

    doi: 10.15252/msb.20166796

    Figure Lengend Snippet: Vemurafenib‐induced de‐differentiation of cells and adaptive resistance are reversible upon drug removal Schematic outline of an experiment involving induction of the slowly cycling NGFR High state in COLO858 cells following 48‐h treatment with 0.32 μM vemurafenib, sorting cells to obtain NGFR Low and NGFR High subpopulations, recovering each cell subpopulation in fresh growth medium for 1–9 days, and re‐inducing recovered cells with vemurafenib. NGFR and Ki‐67 protein levels measured by immunofluorescence in cells grown for 9 days in fresh medium ( n = 4). Growth rate (GR) inhibition assay performed on FACS‐sorted NGFR High and NGFR Low pools of cells after 2 (C) or 9 (D) days of outgrowth in fresh medium. Measurements were performed in 4 (C) or 6 (D) replicates. Growth rate and doubling time measurements in 4 replicates in FACS‐sorted NGFR High and NGFR Low cells during 2–5 days of outgrowth in fresh medium. NGFR levels measured in duplicate by immunofluorescence in COLO858 cells recovered after 9 days of outgrowth in fresh media and subsequently re‐exposed for 48 h to four doses of vemurafenib. Data information: Data in (B–F) are presented as mean ± SD. Statistical significance was determined by two‐way ANOVA.

    Article Snippet: Sections were incubated for 30 min with primary antibodies including Ki‐67 rabbit monoclonal antibody (clone SP6, Cat# VP‐RM04) from Vector Laboratories, p75NTR/NGFR rabbit polyclonal antibody (Cat# 119‐11668) from RayBiotech, and MITF mouse monoclonal antibody (clone D5, Cat# MA5‐14154) from Thermo Scientific, and were then completed with the Leica Refine detection kit (secondary antibody, the DAB chromogen, and the hematoxylin counterstain).

    Techniques: Immunofluorescence, Inhibition, FACS

    The NGFR High state is associated with resistance to MAPK inhibitors in a subset of melanoma patients Immunohistochemical analysis of vemurafenib‐naïve tumors from three melanoma patients stained for NGFR, MITF, and Ki‐67 (see Materials and Methods for patient clinical information). Covariate single‐cell analysis of Ki‐67 versus NGFR measured by immunofluorescence in pre‐treatment, on‐treatment (with dabrafenib and trametinib combination for 2 weeks), and post‐relapse tumor biopsies of a BRAF ‐mutant melanoma patient (see Materials and Methods for patient clinical information). Cell population histograms representing c‐Jun variations measured by immunofluorescence in the same patient‐matched biopsies as shown in (B). NGFR gene expression changes in 21 matched pairs of pre‐treatment and post‐resistance tumor biopsies analyzed by RNA sequencing. MITF changes are shown for tumors with a post‐resistance NGFR increase (increase = log 2 (fold‐change) > 0.5, decrease = log 2 (fold‐change)

    Journal: Molecular Systems Biology

    Article Title: Adaptive resistance of melanoma cells to RAF inhibition via reversible induction of a slowly dividing de‐differentiated state

    doi: 10.15252/msb.20166796

    Figure Lengend Snippet: The NGFR High state is associated with resistance to MAPK inhibitors in a subset of melanoma patients Immunohistochemical analysis of vemurafenib‐naïve tumors from three melanoma patients stained for NGFR, MITF, and Ki‐67 (see Materials and Methods for patient clinical information). Covariate single‐cell analysis of Ki‐67 versus NGFR measured by immunofluorescence in pre‐treatment, on‐treatment (with dabrafenib and trametinib combination for 2 weeks), and post‐relapse tumor biopsies of a BRAF ‐mutant melanoma patient (see Materials and Methods for patient clinical information). Cell population histograms representing c‐Jun variations measured by immunofluorescence in the same patient‐matched biopsies as shown in (B). NGFR gene expression changes in 21 matched pairs of pre‐treatment and post‐resistance tumor biopsies analyzed by RNA sequencing. MITF changes are shown for tumors with a post‐resistance NGFR increase (increase = log 2 (fold‐change) > 0.5, decrease = log 2 (fold‐change)

    Article Snippet: Sections were incubated for 30 min with primary antibodies including Ki‐67 rabbit monoclonal antibody (clone SP6, Cat# VP‐RM04) from Vector Laboratories, p75NTR/NGFR rabbit polyclonal antibody (Cat# 119‐11668) from RayBiotech, and MITF mouse monoclonal antibody (clone D5, Cat# MA5‐14154) from Thermo Scientific, and were then completed with the Leica Refine detection kit (secondary antibody, the DAB chromogen, and the hematoxylin counterstain).

    Techniques: Immunohistochemistry, Staining, Single-cell Analysis, Immunofluorescence, Mutagenesis, Expressing, RNA Sequencing Assay

    The CPC and CENP-E exhibit distinct central spindle transfer mechanisms during anaphase. (A–F) CENP-E transfers to the central spindle at anaphase even when the CPC remains stuck on the chromatin. CENP-E (green; panels 3 and 7) and α-tubulin or TrAP-INCENP (red; panels 2 and 6) plus DAPI for DNA (blue; panels 4 and 8) are shown for INCENP ON cells (A), INCENP OFF cells (B), or INCENP OFF cells expressing TrAP-INCENP WT (C), TrAP-INCENP W766G (D), TrAP-INCENP F802A (E), or TrAP-INCENP WT in the presence of ZM447439 (F). CENP-E localizes to the outer kinetochore in all cells (insets) and transfers to the central spindle at anaphase, even in cells expressing TrAP-INCENP W766G or TrAP-INCENP F802A in which the CPC remains trapped on the chromatin. Insets show magnified views of boxed regions. Bars, 5 µm. (G) The level of H3S10ph is reduced significantly in the presence of ZM447439 as a result of aurora B inhibition. Levels of aurora B and PP1 remained the same. α-Tubulin was used as a loading control.

    Journal: The Journal of Cell Biology

    Article Title: INCENP-aurora B interactions modulate kinase activity and chromosome passenger complex localization

    doi: 10.1083/jcb.200906053

    Figure Lengend Snippet: The CPC and CENP-E exhibit distinct central spindle transfer mechanisms during anaphase. (A–F) CENP-E transfers to the central spindle at anaphase even when the CPC remains stuck on the chromatin. CENP-E (green; panels 3 and 7) and α-tubulin or TrAP-INCENP (red; panels 2 and 6) plus DAPI for DNA (blue; panels 4 and 8) are shown for INCENP ON cells (A), INCENP OFF cells (B), or INCENP OFF cells expressing TrAP-INCENP WT (C), TrAP-INCENP W766G (D), TrAP-INCENP F802A (E), or TrAP-INCENP WT in the presence of ZM447439 (F). CENP-E localizes to the outer kinetochore in all cells (insets) and transfers to the central spindle at anaphase, even in cells expressing TrAP-INCENP W766G or TrAP-INCENP F802A in which the CPC remains trapped on the chromatin. Insets show magnified views of boxed regions. Bars, 5 µm. (G) The level of H3S10ph is reduced significantly in the presence of ZM447439 as a result of aurora B inhibition. Levels of aurora B and PP1 remained the same. α-Tubulin was used as a loading control.

    Article Snippet: Anti–α-tubulin antibody (B512; Sigma-Aldrich), anti-H3S10ph (Millipore), and anti-H3S28ph (Abcam) were used.

    Techniques: Expressing, Inhibition

    Phenotypic analysis of conditional INCENP knockout cell line. (A) Immunofluorescence images showing INCENP ON and INCENP OFF cells stained with specific antibodies recognizing endogenous INCENP (green; panels 3 and 7), α-tubulin (red; panels 2 and 6), and DAPI for DNA (blue; panels 4 and 8). After incubation with doxycycline for 26 h, endogenous INCENP was hardly detected (INCENP OFF ; panel 7). Panels 9–16 show immunofluorescence images of H3S10ph (green; panels 10 and 14) and H3S28ph (red; panels 11 and 15) plus DAPI for DNA (blue; panels 12 and16) in INCENP ON (panels 9–12) and INCENP OFF (panels 13–16) cells. Merged images are shown in panels 1, 5, 9, and 13. INCENP ON and INCENP OFF cells were harvested and fixed at the same time. Images were acquired using the same microscope settings for all experiments. Bar, 5 µm. (B) Mitotic indices of wild-type, INCENP ON , and INCENP OFF cells. (C) Scoring of mitotic cells in late prometaphase and metaphase where cells are bioriented and two spindle poles are on opposite sides. (D–G) Percentage of mitotic cells in prometaphase or metaphase (D), anaphase (E), telophase (F), or cytokinesis (G) is shown. (H–J) Percentage of binucleated cells (H), multinucleated cells (I), and mitotic cells with multipolar spindles (J) is shown. Error bars indicate SD.

    Journal: The Journal of Cell Biology

    Article Title: INCENP-aurora B interactions modulate kinase activity and chromosome passenger complex localization

    doi: 10.1083/jcb.200906053

    Figure Lengend Snippet: Phenotypic analysis of conditional INCENP knockout cell line. (A) Immunofluorescence images showing INCENP ON and INCENP OFF cells stained with specific antibodies recognizing endogenous INCENP (green; panels 3 and 7), α-tubulin (red; panels 2 and 6), and DAPI for DNA (blue; panels 4 and 8). After incubation with doxycycline for 26 h, endogenous INCENP was hardly detected (INCENP OFF ; panel 7). Panels 9–16 show immunofluorescence images of H3S10ph (green; panels 10 and 14) and H3S28ph (red; panels 11 and 15) plus DAPI for DNA (blue; panels 12 and16) in INCENP ON (panels 9–12) and INCENP OFF (panels 13–16) cells. Merged images are shown in panels 1, 5, 9, and 13. INCENP ON and INCENP OFF cells were harvested and fixed at the same time. Images were acquired using the same microscope settings for all experiments. Bar, 5 µm. (B) Mitotic indices of wild-type, INCENP ON , and INCENP OFF cells. (C) Scoring of mitotic cells in late prometaphase and metaphase where cells are bioriented and two spindle poles are on opposite sides. (D–G) Percentage of mitotic cells in prometaphase or metaphase (D), anaphase (E), telophase (F), or cytokinesis (G) is shown. (H–J) Percentage of binucleated cells (H), multinucleated cells (I), and mitotic cells with multipolar spindles (J) is shown. Error bars indicate SD.

    Article Snippet: Anti–α-tubulin antibody (B512; Sigma-Aldrich), anti-H3S10ph (Millipore), and anti-H3S28ph (Abcam) were used.

    Techniques: Knock-Out, Immunofluorescence, Staining, Incubation, Microscopy

    Kinetochore assembly is normal in cells lacking a functional CPC. (A–E) Kinetochore proteins CENP-A (A), CENP-H (B), CENP-O (C), CENP-T (D), and Hec1 (E) localize normally during metaphase and anaphase in INCENP ON cells (panels 1–8) and INCENP OFF cells (panels 9–16). CENP proteins are shown in green, α-tubulin or CENP-H–GFP in red, and DAPI (DNA) in blue. Insets show magnified views of boxed regions. Bars, 5 µm.

    Journal: The Journal of Cell Biology

    Article Title: INCENP-aurora B interactions modulate kinase activity and chromosome passenger complex localization

    doi: 10.1083/jcb.200906053

    Figure Lengend Snippet: Kinetochore assembly is normal in cells lacking a functional CPC. (A–E) Kinetochore proteins CENP-A (A), CENP-H (B), CENP-O (C), CENP-T (D), and Hec1 (E) localize normally during metaphase and anaphase in INCENP ON cells (panels 1–8) and INCENP OFF cells (panels 9–16). CENP proteins are shown in green, α-tubulin or CENP-H–GFP in red, and DAPI (DNA) in blue. Insets show magnified views of boxed regions. Bars, 5 µm.

    Article Snippet: Anti–α-tubulin antibody (B512; Sigma-Aldrich), anti-H3S10ph (Millipore), and anti-H3S28ph (Abcam) were used.

    Techniques: Functional Assay

    Quantitation of aurora B kinase activity in INCENP mutants. (A) Estimation of aurora B activity by immunoblotting. Asynchronous cells were harvested after treatment with doxycycline for 28 h or with 2 µM ZM447439 for 5 h ( Fig. S1 C ), and lysates were subjected to immunoblotting with the indicated antibodies. α-Tubulin and haspin kinase substrate H3T3ph are shown as controls. White lines indicate that intervening lanes have been spliced out. (B) Measurement of H3S10ph levels in the same samples using Odyssey. (C) Ratio of aurora B protein levels versus the loading control α-tubulin for each sample as measured using Odyssey. (D) Mitotic index of each cell line at the time of harvesting. (E) Diagram showing the relative levels of aurora B activity based on the level of H3S10ph measured using the Odyssey assay. WT, wild type. Error bars indicate SD.

    Journal: The Journal of Cell Biology

    Article Title: INCENP-aurora B interactions modulate kinase activity and chromosome passenger complex localization

    doi: 10.1083/jcb.200906053

    Figure Lengend Snippet: Quantitation of aurora B kinase activity in INCENP mutants. (A) Estimation of aurora B activity by immunoblotting. Asynchronous cells were harvested after treatment with doxycycline for 28 h or with 2 µM ZM447439 for 5 h ( Fig. S1 C ), and lysates were subjected to immunoblotting with the indicated antibodies. α-Tubulin and haspin kinase substrate H3T3ph are shown as controls. White lines indicate that intervening lanes have been spliced out. (B) Measurement of H3S10ph levels in the same samples using Odyssey. (C) Ratio of aurora B protein levels versus the loading control α-tubulin for each sample as measured using Odyssey. (D) Mitotic index of each cell line at the time of harvesting. (E) Diagram showing the relative levels of aurora B activity based on the level of H3S10ph measured using the Odyssey assay. WT, wild type. Error bars indicate SD.

    Article Snippet: Anti–α-tubulin antibody (B512; Sigma-Aldrich), anti-H3S10ph (Millipore), and anti-H3S28ph (Abcam) were used.

    Techniques: Quantitation Assay, Activity Assay

    Generation of INCENP structural mutants to dissect INCENP–aurora B interactions. (A) Schematic representation of the main domains of INCENP with a sequence alignment of the INCENP IN box in several model organisms. Red triangles indicate key residues mutated in this study. Xl, Xenopus laevis ; Gg, Gallus gallus ; Hs, Homo sapiens ; Mm, Mus musculus ; Dm, Drosophila melanogaster ; Ce, Caenorhabditis elegans ; Sc, Saccharomyces cerevisiae . (B–D) Growth curves of DT40 wild-type (WT) and INCENP ON/OFF cells expressing various forms of INCENP in the presence and absence of doxycycline. The expressed INCENP was TrAP-INCENP WT (B), TrAP-INCENP WT plus TrAP-INCENP W766G (C), and TrAP-INCENP WT plus TrAP-INCENP F802A (D). Growth of cultures expressing TrAP-INCENP WT was indistinguishable from wild-type cells. All cultures expressing only mutant INCENP died. (E–G) Immunoblots show levels of chromosomal passenger proteins in INCENP ON/OFF cells (E) or INCENP ON/OFF cells expressing TrAP-INCENP W766G (F) or TrAP-INCENP F802A (G). INCENP OFF cells expressing TrAP-INCENP WT are shown as controls. (E) Black circles indicate a proteolytic fragment of INCENP. At time 0, INCENP, which was driven by the tTA expressed from the KIF4A promoter, is ∼20× overexpressed, and the levels of the other passengers are correspondingly increased. All levels decrease to those shown in wild-type clone 18 cells after the addition of doxycycline provided that some form of INCENP was present. Levels of H3S10ph are shown as a measure of aurora B activity and α-tubulin as a loading control. White lines indicate that intervening lanes have been spliced out. (H) Immunoblots shows higher resolutions of INCENP and its fragment. TrAP-INCENP class I comigrate with INCENP class II. The red line indicates that the proteolytic fragments seen in all cells expressing TrAP-INCENP constructs are not INCENP class I. α-Tubulin was used as a loading control. (F–H) Black circles indicate the position of INCENP degradation fragments. Error bars indicate SD.

    Journal: The Journal of Cell Biology

    Article Title: INCENP-aurora B interactions modulate kinase activity and chromosome passenger complex localization

    doi: 10.1083/jcb.200906053

    Figure Lengend Snippet: Generation of INCENP structural mutants to dissect INCENP–aurora B interactions. (A) Schematic representation of the main domains of INCENP with a sequence alignment of the INCENP IN box in several model organisms. Red triangles indicate key residues mutated in this study. Xl, Xenopus laevis ; Gg, Gallus gallus ; Hs, Homo sapiens ; Mm, Mus musculus ; Dm, Drosophila melanogaster ; Ce, Caenorhabditis elegans ; Sc, Saccharomyces cerevisiae . (B–D) Growth curves of DT40 wild-type (WT) and INCENP ON/OFF cells expressing various forms of INCENP in the presence and absence of doxycycline. The expressed INCENP was TrAP-INCENP WT (B), TrAP-INCENP WT plus TrAP-INCENP W766G (C), and TrAP-INCENP WT plus TrAP-INCENP F802A (D). Growth of cultures expressing TrAP-INCENP WT was indistinguishable from wild-type cells. All cultures expressing only mutant INCENP died. (E–G) Immunoblots show levels of chromosomal passenger proteins in INCENP ON/OFF cells (E) or INCENP ON/OFF cells expressing TrAP-INCENP W766G (F) or TrAP-INCENP F802A (G). INCENP OFF cells expressing TrAP-INCENP WT are shown as controls. (E) Black circles indicate a proteolytic fragment of INCENP. At time 0, INCENP, which was driven by the tTA expressed from the KIF4A promoter, is ∼20× overexpressed, and the levels of the other passengers are correspondingly increased. All levels decrease to those shown in wild-type clone 18 cells after the addition of doxycycline provided that some form of INCENP was present. Levels of H3S10ph are shown as a measure of aurora B activity and α-tubulin as a loading control. White lines indicate that intervening lanes have been spliced out. (H) Immunoblots shows higher resolutions of INCENP and its fragment. TrAP-INCENP class I comigrate with INCENP class II. The red line indicates that the proteolytic fragments seen in all cells expressing TrAP-INCENP constructs are not INCENP class I. α-Tubulin was used as a loading control. (F–H) Black circles indicate the position of INCENP degradation fragments. Error bars indicate SD.

    Article Snippet: Anti–α-tubulin antibody (B512; Sigma-Aldrich), anti-H3S10ph (Millipore), and anti-H3S28ph (Abcam) were used.

    Techniques: Sequencing, Expressing, Mutagenesis, Western Blot, Activity Assay, Construct

    In vivo analysis of CPC formation. (A and B) INCENP ON/OFF cells stably expressing TrAP-tagged INCENP mutants were grown in doxycycline (Dox) to shut off expression of wild-type (WT) INCENP plus nocodazole (Noc; or not) to enrich for mitotic cells. (A) Immunoblots of streptavidin pull-downs with anti-SBP to reveal the proteins associated with INCENP. (B) Immunoblots of whole cell lysates with antibodies to INCENP (monoclonal antibody 3D3; Cooke et al., 1987 ) and the other passenger proteins. Equal numbers of cells were loaded per lane. α-Tubulin is used as a loading control. White lines indicate that intervening lanes have been spliced out. (C–H) Localization of exogenous TrAP-INCENP (red; panel 2) plus endogenous aurora B (green; C, E, G, and I [panel 3]) or CENP-A (green; D, F, H, and J [panel 3]) in INCENP OFF cells expressing TrAP-INCENP WT (C and D), TrAP-INCENP W766G (E and F), or TrAP-INCENP F802A (G and H). These images are of the nocodazole-treated cells used for the SBP pull-down experiment in A and B. (E) In all cases, the TrAP-INCENP localizes to centromeres, but only in INCENP OFF /TrAP-INCENP W766G cells is aurora B localization diffuse. Images were acquired using the same microscope settings for all experiments. Insets show magnified views of boxed regions. Bars, 5 µm.

    Journal: The Journal of Cell Biology

    Article Title: INCENP-aurora B interactions modulate kinase activity and chromosome passenger complex localization

    doi: 10.1083/jcb.200906053

    Figure Lengend Snippet: In vivo analysis of CPC formation. (A and B) INCENP ON/OFF cells stably expressing TrAP-tagged INCENP mutants were grown in doxycycline (Dox) to shut off expression of wild-type (WT) INCENP plus nocodazole (Noc; or not) to enrich for mitotic cells. (A) Immunoblots of streptavidin pull-downs with anti-SBP to reveal the proteins associated with INCENP. (B) Immunoblots of whole cell lysates with antibodies to INCENP (monoclonal antibody 3D3; Cooke et al., 1987 ) and the other passenger proteins. Equal numbers of cells were loaded per lane. α-Tubulin is used as a loading control. White lines indicate that intervening lanes have been spliced out. (C–H) Localization of exogenous TrAP-INCENP (red; panel 2) plus endogenous aurora B (green; C, E, G, and I [panel 3]) or CENP-A (green; D, F, H, and J [panel 3]) in INCENP OFF cells expressing TrAP-INCENP WT (C and D), TrAP-INCENP W766G (E and F), or TrAP-INCENP F802A (G and H). These images are of the nocodazole-treated cells used for the SBP pull-down experiment in A and B. (E) In all cases, the TrAP-INCENP localizes to centromeres, but only in INCENP OFF /TrAP-INCENP W766G cells is aurora B localization diffuse. Images were acquired using the same microscope settings for all experiments. Insets show magnified views of boxed regions. Bars, 5 µm.

    Article Snippet: Anti–α-tubulin antibody (B512; Sigma-Aldrich), anti-H3S10ph (Millipore), and anti-H3S28ph (Abcam) were used.

    Techniques: In Vivo, Stable Transfection, Expressing, Western Blot, Microscopy

    Generation and characterization of an INCENP promoter hijack–conditional knockout cell line. (A) Map of the chicken INCENP locus showing the region replaced in the promoter hijack, the size of the resulting restriction fragments, and the probe used in Southern analysis (red box). (B) Southern analysis showing the first targeting (PuroR) and the digestion after removal of the puromycin marker (PuroS; Samejima et al., 2008 ). (C) Map of the chicken INCENP locus showing the region targeted by the gene disruption construct. (D) Southern analysis demonstrating targeting of the gene disruption construct. Only one band is observed because the probe (red bar) recognizes a region deleted in the promoter hijack of the first allele. KO, knockout. (E) Statistical summary of the targeting efficiencies. (F) Relative mRNA level of incenp and incenp-like gene expressed in wild-type clone 18 cells. Three independent RNA preparations were used in three independent experiments. (G) Growth curves of DT40 wild-type (WT; clone 18), INCENP ON , and INCENP OFF cells. Wild-type and INCENP ON cells have a similar proliferation rate. INCENP OFF cells cease proliferation 12 h after doxycycline addition. (H) Immunoblot showing that endogenous INCENP is efficiently depleted by 20 h after the addition of doxycycline. α-Tubulin was used as a loading control. (I) INCENP OFF cells are defective in completion of chromosome alignment. Live cell imaging of INCENP ON/OFF cells stably expressing histone H2BRFP revealed that INCENP OFF cells stay for a statistically longer period in prometaphase before entering to anaphase. Images were taken every 2 min. (B and D) X indicates the promoter hijack allele. Error bars indicate SD.

    Journal: The Journal of Cell Biology

    Article Title: INCENP-aurora B interactions modulate kinase activity and chromosome passenger complex localization

    doi: 10.1083/jcb.200906053

    Figure Lengend Snippet: Generation and characterization of an INCENP promoter hijack–conditional knockout cell line. (A) Map of the chicken INCENP locus showing the region replaced in the promoter hijack, the size of the resulting restriction fragments, and the probe used in Southern analysis (red box). (B) Southern analysis showing the first targeting (PuroR) and the digestion after removal of the puromycin marker (PuroS; Samejima et al., 2008 ). (C) Map of the chicken INCENP locus showing the region targeted by the gene disruption construct. (D) Southern analysis demonstrating targeting of the gene disruption construct. Only one band is observed because the probe (red bar) recognizes a region deleted in the promoter hijack of the first allele. KO, knockout. (E) Statistical summary of the targeting efficiencies. (F) Relative mRNA level of incenp and incenp-like gene expressed in wild-type clone 18 cells. Three independent RNA preparations were used in three independent experiments. (G) Growth curves of DT40 wild-type (WT; clone 18), INCENP ON , and INCENP OFF cells. Wild-type and INCENP ON cells have a similar proliferation rate. INCENP OFF cells cease proliferation 12 h after doxycycline addition. (H) Immunoblot showing that endogenous INCENP is efficiently depleted by 20 h after the addition of doxycycline. α-Tubulin was used as a loading control. (I) INCENP OFF cells are defective in completion of chromosome alignment. Live cell imaging of INCENP ON/OFF cells stably expressing histone H2BRFP revealed that INCENP OFF cells stay for a statistically longer period in prometaphase before entering to anaphase. Images were taken every 2 min. (B and D) X indicates the promoter hijack allele. Error bars indicate SD.

    Article Snippet: Anti–α-tubulin antibody (B512; Sigma-Aldrich), anti-H3S10ph (Millipore), and anti-H3S28ph (Abcam) were used.

    Techniques: Knock-Out, Marker, Construct, Gene Knockout, Live Cell Imaging, Stable Transfection, Expressing

    Active aurora B is required for aurora B and INCENP to transfer to the spindle midzone. (A–C) Localization of exogenous TrAP-INCENP (red; panels 2, 6, 10, and 14) plus endogenous aurora B (green; panels 3 and 7) or CENP-A (green; panels 11 and 15) and DNA (blue; panels 4, 8, 12, and 16) in INCENP OFF cells expressing TrAP-INCENP WT (A), TrAP-INCENP W766G (B), or TrAP-INCENP F802A (C). In all cases, TrAP-INCENP localizes to centromeres at metaphase, but in INCENP OFF cells expressing TrAP-INCENP W766G or TrAP-INCENP F802A (B and C), it fails to transfer to the spindle midzone at anaphase. Aurora B colocalizes with INCENP except for cells expressing TrAP-INCENP W766G , where it is diffuse. (D and E) INCENP and aurora B localize to centromeres in early mitosis but fail to transfer to the spindle at anaphase in wild-type clone 18 cells grown in the aurora B inhibitor ZM447439. Cells were stained for INCENP or aurora B (green; panels 3, 7, and 11) plus α-tubulin (red; panels 2, 6, and 10) and DAPI for DNA (blue; panels 4, 8, and 12). Insets show magnified views of boxed regions. Bars, 5 µm.

    Journal: The Journal of Cell Biology

    Article Title: INCENP-aurora B interactions modulate kinase activity and chromosome passenger complex localization

    doi: 10.1083/jcb.200906053

    Figure Lengend Snippet: Active aurora B is required for aurora B and INCENP to transfer to the spindle midzone. (A–C) Localization of exogenous TrAP-INCENP (red; panels 2, 6, 10, and 14) plus endogenous aurora B (green; panels 3 and 7) or CENP-A (green; panels 11 and 15) and DNA (blue; panels 4, 8, 12, and 16) in INCENP OFF cells expressing TrAP-INCENP WT (A), TrAP-INCENP W766G (B), or TrAP-INCENP F802A (C). In all cases, TrAP-INCENP localizes to centromeres at metaphase, but in INCENP OFF cells expressing TrAP-INCENP W766G or TrAP-INCENP F802A (B and C), it fails to transfer to the spindle midzone at anaphase. Aurora B colocalizes with INCENP except for cells expressing TrAP-INCENP W766G , where it is diffuse. (D and E) INCENP and aurora B localize to centromeres in early mitosis but fail to transfer to the spindle at anaphase in wild-type clone 18 cells grown in the aurora B inhibitor ZM447439. Cells were stained for INCENP or aurora B (green; panels 3, 7, and 11) plus α-tubulin (red; panels 2, 6, and 10) and DAPI for DNA (blue; panels 4, 8, and 12). Insets show magnified views of boxed regions. Bars, 5 µm.

    Article Snippet: Anti–α-tubulin antibody (B512; Sigma-Aldrich), anti-H3S10ph (Millipore), and anti-H3S28ph (Abcam) were used.

    Techniques: Expressing, Staining

    A model for defective huntingtin-mediated cofilin rod stress response leading to activation of TG2. The grey arrow pathway highlights normal huntingtin stress response by releasing from the ER, entering the nucleus and binding cofilin–actin rods, then exiting the nucleus upon stress relief. With mutant huntingtin present, the dashed arrow pathways show a defect in huntingtin stress response, resulting in less nuclear activity and persistent rods. Back in the cytoplasm, the black arrow pathways highlight elevated calcium due to a defective ER, which results in aberrant TG2 activation and cross-linking of cofilin–actin in both the nucleus and cytoplasm. Defective actin remodeling critically affects neurons at the level of dendritic and synaptic dysfunction, as well as exocytosis activity in peripheral cells.

    Journal: Human Molecular Genetics

    Article Title: Mutant huntingtin causes defective actin remodeling during stress: defining a new role for transglutaminase 2 in neurodegenerative disease

    doi: 10.1093/hmg/ddr075

    Figure Lengend Snippet: A model for defective huntingtin-mediated cofilin rod stress response leading to activation of TG2. The grey arrow pathway highlights normal huntingtin stress response by releasing from the ER, entering the nucleus and binding cofilin–actin rods, then exiting the nucleus upon stress relief. With mutant huntingtin present, the dashed arrow pathways show a defect in huntingtin stress response, resulting in less nuclear activity and persistent rods. Back in the cytoplasm, the black arrow pathways highlight elevated calcium due to a defective ER, which results in aberrant TG2 activation and cross-linking of cofilin–actin in both the nucleus and cytoplasm. Defective actin remodeling critically affects neurons at the level of dendritic and synaptic dysfunction, as well as exocytosis activity in peripheral cells.

    Article Snippet: Primary antibodies include: affinity-purified rabbit IgG to chick actin depolymerizing factor (ADF) (2 ng/l rabbit 1439), which cross-reacts with mammalian ADF and cofilin, and MAB2166 ant huntingtin (Millipore; 1:50 dilution), which were both diluted in 2% FBS in PBS plus 0.05% Tween-20 (PBST) and incubated with the cells overnight at 4°C.

    Techniques: Activation Assay, Binding Assay, Mutagenesis, Activity Assay

    TG2 directly interacts with cofilin–actin rods during stress and TG2 over-expression induces a cofilin–actin complex in stressed cells. ( A ) STHdh cells were transiently transfected with mCerulean–cofilin and either eYFP alone (a–c, g–j) or eYFP-TG2 (d–f, k–r), and FLIM analysis was performed either before (a–c, d–f) or following a 45 min heat shock at 42.5°C (g–j, k–n, o–r). Fluorescence lifetimes for mCerulean blue are presented with a continuous pseudocolor rainbow scale representing time values ranging from 1750 to 3250 ps. The lifetime distribution curve of the mCerulean–cofilin is shown as a histogram on the right representing the number of pixels at each lifetime. The red vertical broken line marks the median lifetime distribution for the cell. Red arrows connect histogram value position with lifetime image value. ( B ) Box and whisker plot representing FRET efficiency, with FRET occurring at distances

    Journal: Human Molecular Genetics

    Article Title: Mutant huntingtin causes defective actin remodeling during stress: defining a new role for transglutaminase 2 in neurodegenerative disease

    doi: 10.1093/hmg/ddr075

    Figure Lengend Snippet: TG2 directly interacts with cofilin–actin rods during stress and TG2 over-expression induces a cofilin–actin complex in stressed cells. ( A ) STHdh cells were transiently transfected with mCerulean–cofilin and either eYFP alone (a–c, g–j) or eYFP-TG2 (d–f, k–r), and FLIM analysis was performed either before (a–c, d–f) or following a 45 min heat shock at 42.5°C (g–j, k–n, o–r). Fluorescence lifetimes for mCerulean blue are presented with a continuous pseudocolor rainbow scale representing time values ranging from 1750 to 3250 ps. The lifetime distribution curve of the mCerulean–cofilin is shown as a histogram on the right representing the number of pixels at each lifetime. The red vertical broken line marks the median lifetime distribution for the cell. Red arrows connect histogram value position with lifetime image value. ( B ) Box and whisker plot representing FRET efficiency, with FRET occurring at distances

    Article Snippet: Primary antibodies include: affinity-purified rabbit IgG to chick actin depolymerizing factor (ADF) (2 ng/l rabbit 1439), which cross-reacts with mammalian ADF and cofilin, and MAB2166 ant huntingtin (Millipore; 1:50 dilution), which were both diluted in 2% FBS in PBS plus 0.05% Tween-20 (PBST) and incubated with the cells overnight at 4°C.

    Techniques: Over Expression, Transfection, Fluorescence, Whisker Assay

    Huntingtin is required for normal cell heat shock stress response and mutant huntingtin affects the rate of the nuclear cofilin rod stress response. Temporal imaging in live STHdh Q7/Q7 or STHdh Q111/Q111 cells stably expressing mCerulean–cofilin fusion protein. ( A ) mCerulean–cofilin imaged over time showing nuclear cofilin rod formation in both wild-type and mutant cell lines at 10 and 32 min, respectively (b versus g), following panels show length of times rods exist and time of clearance during maintained heat shock at 42°C (c–e and h–j). ( B ) Comparison graph of average time to nuclear rod formation for stable mCerulean–cofilin wild-type ( N = 15) and mutant ( N = 9) STHdh cells during live cell imaging experiments. * P -value of 0.007. ( C ) Temporal imaging in live STHdh Q7/Q7 cells stably expressing mCerulean–cofilin. Cells were co-transfected with control siRNA or huntingtin-specific siRNA and Block-iT™ Alexa Fluor® red for 72 h prior to experiments. Single-cell visualization of control (a–b) or huntingtin siRNA (h–i) transfection with labeled Block-iT™Alexa Fluor® red. Visualization of STHdh Q7/Q7 cells with mCerulean–cofilin during heat shock, treated with control siRNA (c–g). STHdh Q7/Q7 cells with cofilin–mCerulean during heat shock, treated with siRNA to huntingtin (j–n). ( D ) Comparison graph of average time to cell death in mCerulean–cofilin stable STHdh Q7/Q7 ( N = 13) or STHdh Q111/Q111 ( N = 24) cells imaged live and STHdh Q7/Q7 mCerulean–cofilin cells treated with control siRNA ( N = 5) or huntingtin siRNA ( N = 6) imaged live during heat shock. * P -value of 0.003. Scale bars are 10 µm.

    Journal: Human Molecular Genetics

    Article Title: Mutant huntingtin causes defective actin remodeling during stress: defining a new role for transglutaminase 2 in neurodegenerative disease

    doi: 10.1093/hmg/ddr075

    Figure Lengend Snippet: Huntingtin is required for normal cell heat shock stress response and mutant huntingtin affects the rate of the nuclear cofilin rod stress response. Temporal imaging in live STHdh Q7/Q7 or STHdh Q111/Q111 cells stably expressing mCerulean–cofilin fusion protein. ( A ) mCerulean–cofilin imaged over time showing nuclear cofilin rod formation in both wild-type and mutant cell lines at 10 and 32 min, respectively (b versus g), following panels show length of times rods exist and time of clearance during maintained heat shock at 42°C (c–e and h–j). ( B ) Comparison graph of average time to nuclear rod formation for stable mCerulean–cofilin wild-type ( N = 15) and mutant ( N = 9) STHdh cells during live cell imaging experiments. * P -value of 0.007. ( C ) Temporal imaging in live STHdh Q7/Q7 cells stably expressing mCerulean–cofilin. Cells were co-transfected with control siRNA or huntingtin-specific siRNA and Block-iT™ Alexa Fluor® red for 72 h prior to experiments. Single-cell visualization of control (a–b) or huntingtin siRNA (h–i) transfection with labeled Block-iT™Alexa Fluor® red. Visualization of STHdh Q7/Q7 cells with mCerulean–cofilin during heat shock, treated with control siRNA (c–g). STHdh Q7/Q7 cells with cofilin–mCerulean during heat shock, treated with siRNA to huntingtin (j–n). ( D ) Comparison graph of average time to cell death in mCerulean–cofilin stable STHdh Q7/Q7 ( N = 13) or STHdh Q111/Q111 ( N = 24) cells imaged live and STHdh Q7/Q7 mCerulean–cofilin cells treated with control siRNA ( N = 5) or huntingtin siRNA ( N = 6) imaged live during heat shock. * P -value of 0.003. Scale bars are 10 µm.

    Article Snippet: Primary antibodies include: affinity-purified rabbit IgG to chick actin depolymerizing factor (ADF) (2 ng/l rabbit 1439), which cross-reacts with mammalian ADF and cofilin, and MAB2166 ant huntingtin (Millipore; 1:50 dilution), which were both diluted in 2% FBS in PBS plus 0.05% Tween-20 (PBST) and incubated with the cells overnight at 4°C.

    Techniques: Mutagenesis, Imaging, Stable Transfection, Expressing, Live Cell Imaging, Transfection, Blocking Assay, Labeling

    Full-length, endogenous huntingtin protein is a component of nuclear cofilin–actin stress rods. ( A ) Immunofluorescence in mouse STHdh Q7/Q7 or STHdh Q111/Q111 striatal-derived cell lines, in cells heat-shocked at 42°C for 60 min. Secondary antibodies were either Alexa 488 (green) or Alexa 595 (magenta) labeled. (a–c) Huntingtin monoclonal antibody MAb2166, co-stained with Hoechst DNA dye (cyan). (d–f) Huntingtin monoclonal antibody C20, co-stained with cofilin monoclonal MAb22. (g–i) Huntingtin monoclonal MAb2166 co-stained with an antibody against actin. (j–l) Co-staining with antibodies against cofilin and actin. (m–o) Huntingtin monoclonal antibody MAb2166, in cells expressing an mCerulean–cofilin fusion protein. ( B ) Immunofluorescence on 6-day-old primary hippocampal neurons treated with 10% DMSO for 90 min to induce rod formation. Untreated cell (a–d) stained with affinity purified rabbit IgG to chick ADF (a), huntingtin monoclonal antibody MAb2166 (b) and co-stained with Hoechst DNA dye. Secondary antibodies were either Alexa 488 (green) or Alexa 595 (magenta) labeled. DMSO treated cell (e–h). Merged overlapping signal is pseudo-colored white. Scale bar is 10 µm.

    Journal: Human Molecular Genetics

    Article Title: Mutant huntingtin causes defective actin remodeling during stress: defining a new role for transglutaminase 2 in neurodegenerative disease

    doi: 10.1093/hmg/ddr075

    Figure Lengend Snippet: Full-length, endogenous huntingtin protein is a component of nuclear cofilin–actin stress rods. ( A ) Immunofluorescence in mouse STHdh Q7/Q7 or STHdh Q111/Q111 striatal-derived cell lines, in cells heat-shocked at 42°C for 60 min. Secondary antibodies were either Alexa 488 (green) or Alexa 595 (magenta) labeled. (a–c) Huntingtin monoclonal antibody MAb2166, co-stained with Hoechst DNA dye (cyan). (d–f) Huntingtin monoclonal antibody C20, co-stained with cofilin monoclonal MAb22. (g–i) Huntingtin monoclonal MAb2166 co-stained with an antibody against actin. (j–l) Co-staining with antibodies against cofilin and actin. (m–o) Huntingtin monoclonal antibody MAb2166, in cells expressing an mCerulean–cofilin fusion protein. ( B ) Immunofluorescence on 6-day-old primary hippocampal neurons treated with 10% DMSO for 90 min to induce rod formation. Untreated cell (a–d) stained with affinity purified rabbit IgG to chick ADF (a), huntingtin monoclonal antibody MAb2166 (b) and co-stained with Hoechst DNA dye. Secondary antibodies were either Alexa 488 (green) or Alexa 595 (magenta) labeled. DMSO treated cell (e–h). Merged overlapping signal is pseudo-colored white. Scale bar is 10 µm.

    Article Snippet: Primary antibodies include: affinity-purified rabbit IgG to chick actin depolymerizing factor (ADF) (2 ng/l rabbit 1439), which cross-reacts with mammalian ADF and cofilin, and MAB2166 ant huntingtin (Millipore; 1:50 dilution), which were both diluted in 2% FBS in PBS plus 0.05% Tween-20 (PBST) and incubated with the cells overnight at 4°C.

    Techniques: Immunofluorescence, Derivative Assay, Labeling, Staining, Expressing, Affinity Purification

    Huntingtin protein is required for proper cofilin nuclear rod formation and clearance following cell stress. ( A ) Western blot against huntingtin protein after control siRNA or huntingtin-specific siRNA was expressed in wild-type huntingtin STHdh Q7/Q7 cells for 72 h. Anti-actin shown as a loading control. ( B ) Comparison of percent nuclear rod forming cells with persistent rod phenotype in heat-shocked cells. Comparison between STHdh Q7/Q7 , STHdh Q111/Q111 and STHdh Q7/Q111 cell lines as well as in STHdh Q7/Q7 cells following siRNA treatment. Experiment performed as described previously. N = 3, * P -value of

    Journal: Human Molecular Genetics

    Article Title: Mutant huntingtin causes defective actin remodeling during stress: defining a new role for transglutaminase 2 in neurodegenerative disease

    doi: 10.1093/hmg/ddr075

    Figure Lengend Snippet: Huntingtin protein is required for proper cofilin nuclear rod formation and clearance following cell stress. ( A ) Western blot against huntingtin protein after control siRNA or huntingtin-specific siRNA was expressed in wild-type huntingtin STHdh Q7/Q7 cells for 72 h. Anti-actin shown as a loading control. ( B ) Comparison of percent nuclear rod forming cells with persistent rod phenotype in heat-shocked cells. Comparison between STHdh Q7/Q7 , STHdh Q111/Q111 and STHdh Q7/Q111 cell lines as well as in STHdh Q7/Q7 cells following siRNA treatment. Experiment performed as described previously. N = 3, * P -value of

    Article Snippet: Primary antibodies include: affinity-purified rabbit IgG to chick actin depolymerizing factor (ADF) (2 ng/l rabbit 1439), which cross-reacts with mammalian ADF and cofilin, and MAB2166 ant huntingtin (Millipore; 1:50 dilution), which were both diluted in 2% FBS in PBS plus 0.05% Tween-20 (PBST) and incubated with the cells overnight at 4°C.

    Techniques: Western Blot

    White blood cell populations from HD patients show a cross linked cofilin–actin complex on a western blot which increases with clinical onset and severity of disease. Western blots on the protein extracted from blood buffy coat histopaque-treated pellets from HD patients at different clinically defined stages of HD (the Shoulson–Fahn method). Western blot was done using MAb22 cofilin or anti-actin antibodies. ( A ) Typical comparison of samples between age-matched controls and either pre-symptomatic, early stage 1 and 2 patients and late stage 3 and 4 patients. Mobility of either cofilin or actin is indicated by black arrows. ( B ) Quantification of percent cofilin signal in higher order cofilin–actin band from western blots performed on control and HD patient blood samples. Analysis done using pixel intensity analysis using NIH Image J. Control ( N = 7), pre-symptomatic ( N = 11), early stages 1 + 2 ( N = 9), late stages 3 + 4 ( N = 12). * P -value

    Journal: Human Molecular Genetics

    Article Title: Mutant huntingtin causes defective actin remodeling during stress: defining a new role for transglutaminase 2 in neurodegenerative disease

    doi: 10.1093/hmg/ddr075

    Figure Lengend Snippet: White blood cell populations from HD patients show a cross linked cofilin–actin complex on a western blot which increases with clinical onset and severity of disease. Western blots on the protein extracted from blood buffy coat histopaque-treated pellets from HD patients at different clinically defined stages of HD (the Shoulson–Fahn method). Western blot was done using MAb22 cofilin or anti-actin antibodies. ( A ) Typical comparison of samples between age-matched controls and either pre-symptomatic, early stage 1 and 2 patients and late stage 3 and 4 patients. Mobility of either cofilin or actin is indicated by black arrows. ( B ) Quantification of percent cofilin signal in higher order cofilin–actin band from western blots performed on control and HD patient blood samples. Analysis done using pixel intensity analysis using NIH Image J. Control ( N = 7), pre-symptomatic ( N = 11), early stages 1 + 2 ( N = 9), late stages 3 + 4 ( N = 12). * P -value

    Article Snippet: Primary antibodies include: affinity-purified rabbit IgG to chick actin depolymerizing factor (ADF) (2 ng/l rabbit 1439), which cross-reacts with mammalian ADF and cofilin, and MAB2166 ant huntingtin (Millipore; 1:50 dilution), which were both diluted in 2% FBS in PBS plus 0.05% Tween-20 (PBST) and incubated with the cells overnight at 4°C.

    Techniques: Western Blot

    Mutant huntingtin protein affects nuclear cofilin rod formation and induces persistence of cofilin rods. Immunofluorescence with either huntingtin or cofilin antibodies in either STHdh Q7/Q7 or STHdh Q111/Q111 cell lines. ( A ) Mutant huntingtin protein affects the number and size of nuclear cofilin stress rods upon 60 min heat shock. (a–c) Huntingtin or cofilin immunofluorescence before (a), or after heat shock (b–c) in STHdh Q7/Q7 cells. (d–f) Huntingtin or cofilin immunofluorescence before (d), or after heat shock (e–f) in STHdh Q111/Q111 cells. (g–i) Huntingtin or cofilin immunofluorescence before (g), or after heat shock (h–i) in NIH 3T3 mouse fibroblast cell line. ( B ). Comparison of number of nuclear rods per cell immediately after 60 min heat shock in STHdh Q111/Q111 or STHdh Q7/Q7 lines. Rod forming cells were imaged as a z-stack and the mass projection was used to count number of rods per nucleus in each cell type. N = 3, n = 10 per replicate, total n = 30. * P -value of n

    Journal: Human Molecular Genetics

    Article Title: Mutant huntingtin causes defective actin remodeling during stress: defining a new role for transglutaminase 2 in neurodegenerative disease

    doi: 10.1093/hmg/ddr075

    Figure Lengend Snippet: Mutant huntingtin protein affects nuclear cofilin rod formation and induces persistence of cofilin rods. Immunofluorescence with either huntingtin or cofilin antibodies in either STHdh Q7/Q7 or STHdh Q111/Q111 cell lines. ( A ) Mutant huntingtin protein affects the number and size of nuclear cofilin stress rods upon 60 min heat shock. (a–c) Huntingtin or cofilin immunofluorescence before (a), or after heat shock (b–c) in STHdh Q7/Q7 cells. (d–f) Huntingtin or cofilin immunofluorescence before (d), or after heat shock (e–f) in STHdh Q111/Q111 cells. (g–i) Huntingtin or cofilin immunofluorescence before (g), or after heat shock (h–i) in NIH 3T3 mouse fibroblast cell line. ( B ). Comparison of number of nuclear rods per cell immediately after 60 min heat shock in STHdh Q111/Q111 or STHdh Q7/Q7 lines. Rod forming cells were imaged as a z-stack and the mass projection was used to count number of rods per nucleus in each cell type. N = 3, n = 10 per replicate, total n = 30. * P -value of n

    Article Snippet: Primary antibodies include: affinity-purified rabbit IgG to chick actin depolymerizing factor (ADF) (2 ng/l rabbit 1439), which cross-reacts with mammalian ADF and cofilin, and MAB2166 ant huntingtin (Millipore; 1:50 dilution), which were both diluted in 2% FBS in PBS plus 0.05% Tween-20 (PBST) and incubated with the cells overnight at 4°C.

    Techniques: Mutagenesis, Immunofluorescence

    COPI complex promotes elongation of autophagosomes. ( a ) HEK293 cells stably expressing GFP-DFCP1 or the parental cell line were pre-treated for 3 h with 3 μg ml −1 BFA (long BFA), starved for 1 h in the presence of BFA and the parental cell line was immunolabelled for WIPI2 or ATG16. Cells were imaged by wide-field microscopy. Short BFA corresponds to non-pre-treated cells. Bar corresponds to 10 μm. ( b ) Values are means±s.e.m. of DFCP1, WIPI2 and ATG16 spots per cell in a , from at least six fields with 5–10 cells each. ( c ) HEK293 cells were transfected with δCOP (siδCOP), β'COP (siβ'COP) or non-targeted (siNT) siRNA, starved, immunolabelled for WIPI2 and imaged by wide-field microscopy. Arrows point at WIPI2 ring-like structures. Bar corresponds to 10 μm. ( d ) Values are means±s.e.m. of WIPI2 rings per cell in c , for 10 different fields with 5–15 cells each. ( e ) HEK293 cells were pre-treated for 3 h with 3 μg ml −1 BFA, starved, immunolabelled for WIPI2 and imaged by wide-field microscopy. Values are means±s.e.m. of WIPI2 rings per cell, for 10 different fields with 5–15 cells each. ( f , g ) HEK293 cells stably expressing GFP-ATG13 ( f ) or GFP-DFCP1 ( g ) were pre-treated for 3 h with 3 μg ml −1 BFA, starved and live-imaged in the presence of BFA by wide-field microscopy. The lifespan of ATG13 and DFCP1 particles was quantitated. Values are means±s.e.m. of ATG13 or DFCP1 particle lifespan, from 60 and 30 montages, respectively. ( h , i ) HEK293 cells expressing stably GFP-ATG13 ( h ) or GFP-DFCP1 ( i ) and transiently CFP-LC3 were pre-treated for 3 h with 3 μg ml −1 BFA, starved and live-imaged in the presence of BFA by wide-field microscopy. The lifespan of ATG13 and DFCP1 particles before the appearance of LC3 was quantitated. Values are means±s.e.m. of ATG13 or DFCP1 particle lifespan before the appearance of LC3, from 20 and 26 montages, respectively. Significance levels were determined with unpaired t -tests. * P =0.05%; ** P =0.01%; *** P =0.001%; **** P =0.0001%.

    Journal: Nature Communications

    Article Title: Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles

    doi: 10.1038/ncomms12420

    Figure Lengend Snippet: COPI complex promotes elongation of autophagosomes. ( a ) HEK293 cells stably expressing GFP-DFCP1 or the parental cell line were pre-treated for 3 h with 3 μg ml −1 BFA (long BFA), starved for 1 h in the presence of BFA and the parental cell line was immunolabelled for WIPI2 or ATG16. Cells were imaged by wide-field microscopy. Short BFA corresponds to non-pre-treated cells. Bar corresponds to 10 μm. ( b ) Values are means±s.e.m. of DFCP1, WIPI2 and ATG16 spots per cell in a , from at least six fields with 5–10 cells each. ( c ) HEK293 cells were transfected with δCOP (siδCOP), β'COP (siβ'COP) or non-targeted (siNT) siRNA, starved, immunolabelled for WIPI2 and imaged by wide-field microscopy. Arrows point at WIPI2 ring-like structures. Bar corresponds to 10 μm. ( d ) Values are means±s.e.m. of WIPI2 rings per cell in c , for 10 different fields with 5–15 cells each. ( e ) HEK293 cells were pre-treated for 3 h with 3 μg ml −1 BFA, starved, immunolabelled for WIPI2 and imaged by wide-field microscopy. Values are means±s.e.m. of WIPI2 rings per cell, for 10 different fields with 5–15 cells each. ( f , g ) HEK293 cells stably expressing GFP-ATG13 ( f ) or GFP-DFCP1 ( g ) were pre-treated for 3 h with 3 μg ml −1 BFA, starved and live-imaged in the presence of BFA by wide-field microscopy. The lifespan of ATG13 and DFCP1 particles was quantitated. Values are means±s.e.m. of ATG13 or DFCP1 particle lifespan, from 60 and 30 montages, respectively. ( h , i ) HEK293 cells expressing stably GFP-ATG13 ( h ) or GFP-DFCP1 ( i ) and transiently CFP-LC3 were pre-treated for 3 h with 3 μg ml −1 BFA, starved and live-imaged in the presence of BFA by wide-field microscopy. The lifespan of ATG13 and DFCP1 particles before the appearance of LC3 was quantitated. Values are means±s.e.m. of ATG13 or DFCP1 particle lifespan before the appearance of LC3, from 20 and 26 montages, respectively. Significance levels were determined with unpaired t -tests. * P =0.05%; ** P =0.01%; *** P =0.001%; **** P =0.0001%.

    Article Snippet: The antibodies used in the course of this work were: mouse anti-ATG13 (Millipore, cat. no. MABC46; immuno-fluorescence (IF) 1:100), rabbit anti-ATG101 (Sigma, cat. no. SAB4200175; IF 1:100), mouse anti-WIPI2 (AbD Serotec, cat. no. MCA5780GA; IF 1:200), rabbit anti-ATG16 (MBL, cat. no. PM040; IF 1:100), mouse anti-GFP (Roche, cat. no. 11814460001; IF 1:100), rabbit anti-GFP (gift of L. Roderick, Department of Cardiovascular Sciences, KU Leuven), goat anti-SEC23 (Santa Cruz, cat. no. sc-12107; IF 1:100), rabbit anti-SEC23IP (Sigma, cat. no. HPA038403; IF 1:100), rabbit and mouse anti-ERGIC53 (Santa Cruz, SC66880 and SC271517; IF 1:100), mouse anti-multi ubiquitin (Cayman Chemicals, cat. no. 14220; IF 1:100), mouse anti-GAPDH (Biogenesis, cat. no. 4699-955; WB 1:10000), rabbit anti-LC3 (Sigma, cat. no L7543; WB 1:2000), rabbit anti-ARCN1 (Sigma, cat. no. HPA037573; WB 1:1000), rabbit anti-COPB2 (Novus Biologicals, cat. no. NB120-2899; WB 1:1000) mouse anti-beta COP (a gift from the late Thomas Kreiss), hamster anti-ATG9 (a gift of S. Tooze, The Francis Crick Institute, London), rabbit anti-ATG9 (Cell Signalling, cat. no. 13509; IF 1:100), rabbit anti-ATG9 (GeneTex, cat. no. GTX128427; IF 1:100), goat anti-mouse fluorescein isothiocyanate (FITC) (Jackson ImmunoResearch, cat. no. 115-095-062; 1:100), goat anti-rabbit FITC (Jackson ImmunoResearch, cat. no. 111-085-045; 1:100), goat anti-mouse tetramethylrhodamine TRITC (Jackson ImmunoResearch, cat. no. 115-025-062; 1:100), goat anti-rabbit TRITC (Jackson ImmunoResearch, cat. no. 115-025-144; 1:100), goat anti-mouse Alexa Fluor 647 (Molecular Probes, A-21236; 1:500-1:1000), goat anti-rabbit Alexa Fluor 647 (Molecular Probes, A-21244; 1:500-1:1000), CF568 goat anti-mouse IgG (Biotium, cat. no. 20301; 1:100) and CF568 goat anti-rabbit IgG (Biotium, cat. no. 20099; 1:100).

    Techniques: Stable Transfection, Expressing, Microscopy, Transfection

    ATG13 targets the ER. ( a – d ) HEK293 cells expressing stably GFP-ATG13 and transiently mCherry-dgk1 (ER marker) were starved, live-imaged by wide-field microscopy, fixed on stage and immunolabelled for ATG13 (secondary antibody conjugated to Alexa Fluor 647). The cells were relocated and imaged by SIM (for mCherry and Alexa Fluor 647) and dSTORM (for Alexa Fluor 647). Montages of representative ATG13 particle formation events from the live-cell imaging step (i), different z stacks from the SIM step (ii) and blow-ups of the ATG13 particles from the super-resolution dSTORM images (iii) are shown. Each figure ( a – d ) corresponds to an independent example. ( e ) HEK293 cells expressing transiently GFP-dgk1 (ER marker) were starved, immunolabelled for GFP and imaged by dSTORM. Two representative examples are shown. Bars in SIM images correspond to 1 μm.

    Journal: Nature Communications

    Article Title: Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles

    doi: 10.1038/ncomms12420

    Figure Lengend Snippet: ATG13 targets the ER. ( a – d ) HEK293 cells expressing stably GFP-ATG13 and transiently mCherry-dgk1 (ER marker) were starved, live-imaged by wide-field microscopy, fixed on stage and immunolabelled for ATG13 (secondary antibody conjugated to Alexa Fluor 647). The cells were relocated and imaged by SIM (for mCherry and Alexa Fluor 647) and dSTORM (for Alexa Fluor 647). Montages of representative ATG13 particle formation events from the live-cell imaging step (i), different z stacks from the SIM step (ii) and blow-ups of the ATG13 particles from the super-resolution dSTORM images (iii) are shown. Each figure ( a – d ) corresponds to an independent example. ( e ) HEK293 cells expressing transiently GFP-dgk1 (ER marker) were starved, immunolabelled for GFP and imaged by dSTORM. Two representative examples are shown. Bars in SIM images correspond to 1 μm.

    Article Snippet: The antibodies used in the course of this work were: mouse anti-ATG13 (Millipore, cat. no. MABC46; immuno-fluorescence (IF) 1:100), rabbit anti-ATG101 (Sigma, cat. no. SAB4200175; IF 1:100), mouse anti-WIPI2 (AbD Serotec, cat. no. MCA5780GA; IF 1:200), rabbit anti-ATG16 (MBL, cat. no. PM040; IF 1:100), mouse anti-GFP (Roche, cat. no. 11814460001; IF 1:100), rabbit anti-GFP (gift of L. Roderick, Department of Cardiovascular Sciences, KU Leuven), goat anti-SEC23 (Santa Cruz, cat. no. sc-12107; IF 1:100), rabbit anti-SEC23IP (Sigma, cat. no. HPA038403; IF 1:100), rabbit and mouse anti-ERGIC53 (Santa Cruz, SC66880 and SC271517; IF 1:100), mouse anti-multi ubiquitin (Cayman Chemicals, cat. no. 14220; IF 1:100), mouse anti-GAPDH (Biogenesis, cat. no. 4699-955; WB 1:10000), rabbit anti-LC3 (Sigma, cat. no L7543; WB 1:2000), rabbit anti-ARCN1 (Sigma, cat. no. HPA037573; WB 1:1000), rabbit anti-COPB2 (Novus Biologicals, cat. no. NB120-2899; WB 1:1000) mouse anti-beta COP (a gift from the late Thomas Kreiss), hamster anti-ATG9 (a gift of S. Tooze, The Francis Crick Institute, London), rabbit anti-ATG9 (Cell Signalling, cat. no. 13509; IF 1:100), rabbit anti-ATG9 (GeneTex, cat. no. GTX128427; IF 1:100), goat anti-mouse fluorescein isothiocyanate (FITC) (Jackson ImmunoResearch, cat. no. 115-095-062; 1:100), goat anti-rabbit FITC (Jackson ImmunoResearch, cat. no. 111-085-045; 1:100), goat anti-mouse tetramethylrhodamine TRITC (Jackson ImmunoResearch, cat. no. 115-025-062; 1:100), goat anti-rabbit TRITC (Jackson ImmunoResearch, cat. no. 115-025-144; 1:100), goat anti-mouse Alexa Fluor 647 (Molecular Probes, A-21236; 1:500-1:1000), goat anti-rabbit Alexa Fluor 647 (Molecular Probes, A-21244; 1:500-1:1000), CF568 goat anti-mouse IgG (Biotium, cat. no. 20301; 1:100) and CF568 goat anti-rabbit IgG (Biotium, cat. no. 20099; 1:100).

    Techniques: Expressing, Stable Transfection, Marker, Microscopy, Live Cell Imaging

    Combination of ERES-ATG9-VMP1 compartments can only partially predict the site of autophagosome nucleation. ( a ) HEK293 cells stably expressing GFP-ATG13 were fed or starved, immunolabelled for ATG9 and ERGIC53 and imaged by wide-field microscopy. Bar corresponds to 10 μm. ( b , c ) Wide-field live-cell imaging of starved HEK293 cells expressing stably GFP-ATG13 and transiently VMP1-mCherry. Representative montages of ATG13 particles forming in association with VMP1 ( b ) or not ( c ) are shown. Arrowheads point at the ATG13 particles in the first two frames from their emergence, the same that were used for the analysis in f . ( d ) HEK293 cells transiently expressing VMP1-YFP were immunolabelled for ubiquitin and imaged by wide-field microscopy. Representative images are shown. Bar corresponds to 10 μm. ( e ) Wide-field live-cell imaging of starved HEK293 cells expressing stably GFP-ATG13 and mRFP-ATG9 and transiently CFP-SEC16 and VMP1-LSS-mKate2. Representative images of a cell expressing the four proteins are shown. Bar corresponds to 10 μm. ( f ) Values are ATG13 particles in b , c associating with VMP1 in the first two frames from their emergence. From analysis of 67 montages. ( g , h ) Values are ATG13 particles associating with any of ATG9, SEC16 and VMP1 ( g ) or with different combinations of them ( h ) in the first two frames from their emergence. Bar in b , c corresponds to 300 nm.

    Journal: Nature Communications

    Article Title: Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles

    doi: 10.1038/ncomms12420

    Figure Lengend Snippet: Combination of ERES-ATG9-VMP1 compartments can only partially predict the site of autophagosome nucleation. ( a ) HEK293 cells stably expressing GFP-ATG13 were fed or starved, immunolabelled for ATG9 and ERGIC53 and imaged by wide-field microscopy. Bar corresponds to 10 μm. ( b , c ) Wide-field live-cell imaging of starved HEK293 cells expressing stably GFP-ATG13 and transiently VMP1-mCherry. Representative montages of ATG13 particles forming in association with VMP1 ( b ) or not ( c ) are shown. Arrowheads point at the ATG13 particles in the first two frames from their emergence, the same that were used for the analysis in f . ( d ) HEK293 cells transiently expressing VMP1-YFP were immunolabelled for ubiquitin and imaged by wide-field microscopy. Representative images are shown. Bar corresponds to 10 μm. ( e ) Wide-field live-cell imaging of starved HEK293 cells expressing stably GFP-ATG13 and mRFP-ATG9 and transiently CFP-SEC16 and VMP1-LSS-mKate2. Representative images of a cell expressing the four proteins are shown. Bar corresponds to 10 μm. ( f ) Values are ATG13 particles in b , c associating with VMP1 in the first two frames from their emergence. From analysis of 67 montages. ( g , h ) Values are ATG13 particles associating with any of ATG9, SEC16 and VMP1 ( g ) or with different combinations of them ( h ) in the first two frames from their emergence. Bar in b , c corresponds to 300 nm.

    Article Snippet: The antibodies used in the course of this work were: mouse anti-ATG13 (Millipore, cat. no. MABC46; immuno-fluorescence (IF) 1:100), rabbit anti-ATG101 (Sigma, cat. no. SAB4200175; IF 1:100), mouse anti-WIPI2 (AbD Serotec, cat. no. MCA5780GA; IF 1:200), rabbit anti-ATG16 (MBL, cat. no. PM040; IF 1:100), mouse anti-GFP (Roche, cat. no. 11814460001; IF 1:100), rabbit anti-GFP (gift of L. Roderick, Department of Cardiovascular Sciences, KU Leuven), goat anti-SEC23 (Santa Cruz, cat. no. sc-12107; IF 1:100), rabbit anti-SEC23IP (Sigma, cat. no. HPA038403; IF 1:100), rabbit and mouse anti-ERGIC53 (Santa Cruz, SC66880 and SC271517; IF 1:100), mouse anti-multi ubiquitin (Cayman Chemicals, cat. no. 14220; IF 1:100), mouse anti-GAPDH (Biogenesis, cat. no. 4699-955; WB 1:10000), rabbit anti-LC3 (Sigma, cat. no L7543; WB 1:2000), rabbit anti-ARCN1 (Sigma, cat. no. HPA037573; WB 1:1000), rabbit anti-COPB2 (Novus Biologicals, cat. no. NB120-2899; WB 1:1000) mouse anti-beta COP (a gift from the late Thomas Kreiss), hamster anti-ATG9 (a gift of S. Tooze, The Francis Crick Institute, London), rabbit anti-ATG9 (Cell Signalling, cat. no. 13509; IF 1:100), rabbit anti-ATG9 (GeneTex, cat. no. GTX128427; IF 1:100), goat anti-mouse fluorescein isothiocyanate (FITC) (Jackson ImmunoResearch, cat. no. 115-095-062; 1:100), goat anti-rabbit FITC (Jackson ImmunoResearch, cat. no. 111-085-045; 1:100), goat anti-mouse tetramethylrhodamine TRITC (Jackson ImmunoResearch, cat. no. 115-025-062; 1:100), goat anti-rabbit TRITC (Jackson ImmunoResearch, cat. no. 115-025-144; 1:100), goat anti-mouse Alexa Fluor 647 (Molecular Probes, A-21236; 1:500-1:1000), goat anti-rabbit Alexa Fluor 647 (Molecular Probes, A-21244; 1:500-1:1000), CF568 goat anti-mouse IgG (Biotium, cat. no. 20301; 1:100) and CF568 goat anti-rabbit IgG (Biotium, cat. no. 20099; 1:100).

    Techniques: Stable Transfection, Expressing, Microscopy, Live Cell Imaging

    ATG9 promotes the formation of ATG13 puncta. ( a ) HEK293 cells were either fed or starved for 1 h, immunolabelled for ATG13 and ATG9 and imaged by wide-field microscopy. Arrowheads in inserts point at ATG13 particles associating with ATG9. Bar corresponds to 10 μm. ( b ) Values are ATG13 particles in c , d associating with ATG9 particles in the first two frames from their emergence. From analysis of 75 montages. ( c , d ) Wide-field live-cell imaging of starved HEK293 cells stably expressing GFP-ATG13 and mRFP-ATG9. Representative montages of ATG13 particles forming in association with ATG9 ( c ) or not ( d ) are shown. Arrowheads point at the ATG13 particles in the first two frames from their emergence, the same that were used for the analysis in b . ( e ) Wide-field live-cell imaging of starved HEK293 cells expressing stably GFP-ATG13 and mRFP-ATG9, and transiently CFP-ER. Representative montage of ATG13 particle forming on a tubular extension of ER previously hosting an ATG9 vesicle is shown. Arrowheads point at the ATG13 particle in the first two frames from its emergence and at the associating extension of ER and ATG9 vesicle. ( f , g ) HEK293 cells were transfected with non-targeted (siNT) or ATG9 (siATG9) siRNA, starved for 1 h, immunolabelled for ATG13 or WIPI2 and imaged by confocal laser scanning microscopy. Values are means±s.e.m. puncta of ATG13 ( f ) or WIPI2 ( g ) per cell, for at least five different fields with 15–30 cells each. Significance levels were determined with unpaired t -tests. Bar in c – e corresponds to 300 nm. ** P =0.01%; *** P =0.001%.

    Journal: Nature Communications

    Article Title: Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles

    doi: 10.1038/ncomms12420

    Figure Lengend Snippet: ATG9 promotes the formation of ATG13 puncta. ( a ) HEK293 cells were either fed or starved for 1 h, immunolabelled for ATG13 and ATG9 and imaged by wide-field microscopy. Arrowheads in inserts point at ATG13 particles associating with ATG9. Bar corresponds to 10 μm. ( b ) Values are ATG13 particles in c , d associating with ATG9 particles in the first two frames from their emergence. From analysis of 75 montages. ( c , d ) Wide-field live-cell imaging of starved HEK293 cells stably expressing GFP-ATG13 and mRFP-ATG9. Representative montages of ATG13 particles forming in association with ATG9 ( c ) or not ( d ) are shown. Arrowheads point at the ATG13 particles in the first two frames from their emergence, the same that were used for the analysis in b . ( e ) Wide-field live-cell imaging of starved HEK293 cells expressing stably GFP-ATG13 and mRFP-ATG9, and transiently CFP-ER. Representative montage of ATG13 particle forming on a tubular extension of ER previously hosting an ATG9 vesicle is shown. Arrowheads point at the ATG13 particle in the first two frames from its emergence and at the associating extension of ER and ATG9 vesicle. ( f , g ) HEK293 cells were transfected with non-targeted (siNT) or ATG9 (siATG9) siRNA, starved for 1 h, immunolabelled for ATG13 or WIPI2 and imaged by confocal laser scanning microscopy. Values are means±s.e.m. puncta of ATG13 ( f ) or WIPI2 ( g ) per cell, for at least five different fields with 15–30 cells each. Significance levels were determined with unpaired t -tests. Bar in c – e corresponds to 300 nm. ** P =0.01%; *** P =0.001%.

    Article Snippet: The antibodies used in the course of this work were: mouse anti-ATG13 (Millipore, cat. no. MABC46; immuno-fluorescence (IF) 1:100), rabbit anti-ATG101 (Sigma, cat. no. SAB4200175; IF 1:100), mouse anti-WIPI2 (AbD Serotec, cat. no. MCA5780GA; IF 1:200), rabbit anti-ATG16 (MBL, cat. no. PM040; IF 1:100), mouse anti-GFP (Roche, cat. no. 11814460001; IF 1:100), rabbit anti-GFP (gift of L. Roderick, Department of Cardiovascular Sciences, KU Leuven), goat anti-SEC23 (Santa Cruz, cat. no. sc-12107; IF 1:100), rabbit anti-SEC23IP (Sigma, cat. no. HPA038403; IF 1:100), rabbit and mouse anti-ERGIC53 (Santa Cruz, SC66880 and SC271517; IF 1:100), mouse anti-multi ubiquitin (Cayman Chemicals, cat. no. 14220; IF 1:100), mouse anti-GAPDH (Biogenesis, cat. no. 4699-955; WB 1:10000), rabbit anti-LC3 (Sigma, cat. no L7543; WB 1:2000), rabbit anti-ARCN1 (Sigma, cat. no. HPA037573; WB 1:1000), rabbit anti-COPB2 (Novus Biologicals, cat. no. NB120-2899; WB 1:1000) mouse anti-beta COP (a gift from the late Thomas Kreiss), hamster anti-ATG9 (a gift of S. Tooze, The Francis Crick Institute, London), rabbit anti-ATG9 (Cell Signalling, cat. no. 13509; IF 1:100), rabbit anti-ATG9 (GeneTex, cat. no. GTX128427; IF 1:100), goat anti-mouse fluorescein isothiocyanate (FITC) (Jackson ImmunoResearch, cat. no. 115-095-062; 1:100), goat anti-rabbit FITC (Jackson ImmunoResearch, cat. no. 111-085-045; 1:100), goat anti-mouse tetramethylrhodamine TRITC (Jackson ImmunoResearch, cat. no. 115-025-062; 1:100), goat anti-rabbit TRITC (Jackson ImmunoResearch, cat. no. 115-025-144; 1:100), goat anti-mouse Alexa Fluor 647 (Molecular Probes, A-21236; 1:500-1:1000), goat anti-rabbit Alexa Fluor 647 (Molecular Probes, A-21244; 1:500-1:1000), CF568 goat anti-mouse IgG (Biotium, cat. no. 20301; 1:100) and CF568 goat anti-rabbit IgG (Biotium, cat. no. 20099; 1:100).

    Techniques: Microscopy, Live Cell Imaging, Stable Transfection, Expressing, Transfection, Confocal Laser Scanning Microscopy

    ATG13 shows unique distribution pattern on autophagosome membranes. ( a ) HEK293 cells were fed or starved in the presence or absence of VPS34 inhibitor for 1 h, immunolabelled for endogenous ATG13 and imaged by dSTORM. The structures of observed ATG13 particles under starved conditions were assigned to four different patterns: crescent shaped (a), semi-spherical (b), quasi-spherical (c) and spherical (d). Representative examples of reconstructed super-resolution images are shown for each pattern. Shapes below the images describe the outline of each observed pattern. Red circles within the spherical pattern correspond to areas of higher density of identified molecules. Values are the percentage of ATG13 particles corresponding to each pattern; from 120 particles analysed. ( b ) HEK293 cells stably expressing GFP-DFCP1 or GFP-ATG13, or the parental cell line were starved, immunolabelled for ATG13, WIPI2, ATG16 or GFP and imaged by dSTORM. Representative examples of reconstructed super-resolution images are shown. Bar corresponds to 0.15 μm. ( c ) The area occupied by ATG13 and ATG16 (labelled with Alexa Fluor 647- or CF 568- conjugated secondary antibody), WIPI2 or GFP-DFCP1 in the reconstructed super-resolution images in b was quantitated. Values are the ratios of ATG13–ATG16, WIPI2 and DFCP1 in each of the analysed particles. Significance levels were determined with one-sample t -test against a theoretical mean of 1 (if ATG13 and ATG16, WIPI2 or DFCP1 occupied the same area), with Bonferroni correction for multiple comparisons. **** P =0.0001%.

    Journal: Nature Communications

    Article Title: Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles

    doi: 10.1038/ncomms12420

    Figure Lengend Snippet: ATG13 shows unique distribution pattern on autophagosome membranes. ( a ) HEK293 cells were fed or starved in the presence or absence of VPS34 inhibitor for 1 h, immunolabelled for endogenous ATG13 and imaged by dSTORM. The structures of observed ATG13 particles under starved conditions were assigned to four different patterns: crescent shaped (a), semi-spherical (b), quasi-spherical (c) and spherical (d). Representative examples of reconstructed super-resolution images are shown for each pattern. Shapes below the images describe the outline of each observed pattern. Red circles within the spherical pattern correspond to areas of higher density of identified molecules. Values are the percentage of ATG13 particles corresponding to each pattern; from 120 particles analysed. ( b ) HEK293 cells stably expressing GFP-DFCP1 or GFP-ATG13, or the parental cell line were starved, immunolabelled for ATG13, WIPI2, ATG16 or GFP and imaged by dSTORM. Representative examples of reconstructed super-resolution images are shown. Bar corresponds to 0.15 μm. ( c ) The area occupied by ATG13 and ATG16 (labelled with Alexa Fluor 647- or CF 568- conjugated secondary antibody), WIPI2 or GFP-DFCP1 in the reconstructed super-resolution images in b was quantitated. Values are the ratios of ATG13–ATG16, WIPI2 and DFCP1 in each of the analysed particles. Significance levels were determined with one-sample t -test against a theoretical mean of 1 (if ATG13 and ATG16, WIPI2 or DFCP1 occupied the same area), with Bonferroni correction for multiple comparisons. **** P =0.0001%.

    Article Snippet: The antibodies used in the course of this work were: mouse anti-ATG13 (Millipore, cat. no. MABC46; immuno-fluorescence (IF) 1:100), rabbit anti-ATG101 (Sigma, cat. no. SAB4200175; IF 1:100), mouse anti-WIPI2 (AbD Serotec, cat. no. MCA5780GA; IF 1:200), rabbit anti-ATG16 (MBL, cat. no. PM040; IF 1:100), mouse anti-GFP (Roche, cat. no. 11814460001; IF 1:100), rabbit anti-GFP (gift of L. Roderick, Department of Cardiovascular Sciences, KU Leuven), goat anti-SEC23 (Santa Cruz, cat. no. sc-12107; IF 1:100), rabbit anti-SEC23IP (Sigma, cat. no. HPA038403; IF 1:100), rabbit and mouse anti-ERGIC53 (Santa Cruz, SC66880 and SC271517; IF 1:100), mouse anti-multi ubiquitin (Cayman Chemicals, cat. no. 14220; IF 1:100), mouse anti-GAPDH (Biogenesis, cat. no. 4699-955; WB 1:10000), rabbit anti-LC3 (Sigma, cat. no L7543; WB 1:2000), rabbit anti-ARCN1 (Sigma, cat. no. HPA037573; WB 1:1000), rabbit anti-COPB2 (Novus Biologicals, cat. no. NB120-2899; WB 1:1000) mouse anti-beta COP (a gift from the late Thomas Kreiss), hamster anti-ATG9 (a gift of S. Tooze, The Francis Crick Institute, London), rabbit anti-ATG9 (Cell Signalling, cat. no. 13509; IF 1:100), rabbit anti-ATG9 (GeneTex, cat. no. GTX128427; IF 1:100), goat anti-mouse fluorescein isothiocyanate (FITC) (Jackson ImmunoResearch, cat. no. 115-095-062; 1:100), goat anti-rabbit FITC (Jackson ImmunoResearch, cat. no. 111-085-045; 1:100), goat anti-mouse tetramethylrhodamine TRITC (Jackson ImmunoResearch, cat. no. 115-025-062; 1:100), goat anti-rabbit TRITC (Jackson ImmunoResearch, cat. no. 115-025-144; 1:100), goat anti-mouse Alexa Fluor 647 (Molecular Probes, A-21236; 1:500-1:1000), goat anti-rabbit Alexa Fluor 647 (Molecular Probes, A-21244; 1:500-1:1000), CF568 goat anti-mouse IgG (Biotium, cat. no. 20301; 1:100) and CF568 goat anti-rabbit IgG (Biotium, cat. no. 20099; 1:100).

    Techniques: Stable Transfection, Expressing

    ER exit promotes the formation of ATG13 puncta. ( a ) HEK293 cells were fed or starved for 1 h, immunolabelled for ATG13 and ERGIC53, and imaged by wide-field microscopy. ( b ) Values are ATG13 particles in c , d associating with SEC16 particles in the first two frames from their emergence. From analysis of 73 montages. ( c , d ) Wide-field live-cell imaging of starved HEK293 cells expressing stably GFP-ATG13 and transiently mCherry-SEC16. Representative montages of ATG13 particles forming in association with SEC16 ( c ) or not ( d ) are shown. Arrowheads point at the ATG13 particles in the first two frames from their emergence, the same that were used for the analysis in b . ( e ) HEK293 cells were fed or starved for 1 h, treated with 50 μM H89 in the last 30 min, immunolabelled for ATG13 and imaged by wide-field microscopy. ( f ) HEK293 cells were starved for 1 h, immunolabelled for βCOP and ERGIC53 and imaged by wide-field microscopy. ( g ) HEK293 cells stably expressing GFP-DFCP1 were fed or starved, immunolabelled for δCOP and imaged by wide-field microscopy. Arrowheads in insets point at COPI particles adjacent to DFCP1 puncta or rings. ( h ) HEK293 cells were pre-treated with 3 μg ml −1 BFA for 3 h (long BFA) or for 30 min (short BFA) and for 30 min with 100 μM FLI06, then starved for 1 h in the presence or absence of BFA and FLI06, immunolabelled for ATG13 and imaged by confocal laser scanning microscopy. ( i ) Values are means±s.e.m. puncta of ATG13 per cell in e , for at least five different fields with 15–30 cells each. ( j ) Values are means±s.e.m. puncta of ATG13 per cell in h , for at least five different fields with 15–30 cells each. ( k ) Values are means±s.e.m. puncta of ATG13 per cell in an independent experiment, for at least five different fields with 15–30 cells each. Significance levels were determined with unpaired t -tests. **** P =0.0001%. Bar in a , e – h corresponds to 10 μm. Bar in c – e corresponds to 300 nm.

    Journal: Nature Communications

    Article Title: Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles

    doi: 10.1038/ncomms12420

    Figure Lengend Snippet: ER exit promotes the formation of ATG13 puncta. ( a ) HEK293 cells were fed or starved for 1 h, immunolabelled for ATG13 and ERGIC53, and imaged by wide-field microscopy. ( b ) Values are ATG13 particles in c , d associating with SEC16 particles in the first two frames from their emergence. From analysis of 73 montages. ( c , d ) Wide-field live-cell imaging of starved HEK293 cells expressing stably GFP-ATG13 and transiently mCherry-SEC16. Representative montages of ATG13 particles forming in association with SEC16 ( c ) or not ( d ) are shown. Arrowheads point at the ATG13 particles in the first two frames from their emergence, the same that were used for the analysis in b . ( e ) HEK293 cells were fed or starved for 1 h, treated with 50 μM H89 in the last 30 min, immunolabelled for ATG13 and imaged by wide-field microscopy. ( f ) HEK293 cells were starved for 1 h, immunolabelled for βCOP and ERGIC53 and imaged by wide-field microscopy. ( g ) HEK293 cells stably expressing GFP-DFCP1 were fed or starved, immunolabelled for δCOP and imaged by wide-field microscopy. Arrowheads in insets point at COPI particles adjacent to DFCP1 puncta or rings. ( h ) HEK293 cells were pre-treated with 3 μg ml −1 BFA for 3 h (long BFA) or for 30 min (short BFA) and for 30 min with 100 μM FLI06, then starved for 1 h in the presence or absence of BFA and FLI06, immunolabelled for ATG13 and imaged by confocal laser scanning microscopy. ( i ) Values are means±s.e.m. puncta of ATG13 per cell in e , for at least five different fields with 15–30 cells each. ( j ) Values are means±s.e.m. puncta of ATG13 per cell in h , for at least five different fields with 15–30 cells each. ( k ) Values are means±s.e.m. puncta of ATG13 per cell in an independent experiment, for at least five different fields with 15–30 cells each. Significance levels were determined with unpaired t -tests. **** P =0.0001%. Bar in a , e – h corresponds to 10 μm. Bar in c – e corresponds to 300 nm.

    Article Snippet: The antibodies used in the course of this work were: mouse anti-ATG13 (Millipore, cat. no. MABC46; immuno-fluorescence (IF) 1:100), rabbit anti-ATG101 (Sigma, cat. no. SAB4200175; IF 1:100), mouse anti-WIPI2 (AbD Serotec, cat. no. MCA5780GA; IF 1:200), rabbit anti-ATG16 (MBL, cat. no. PM040; IF 1:100), mouse anti-GFP (Roche, cat. no. 11814460001; IF 1:100), rabbit anti-GFP (gift of L. Roderick, Department of Cardiovascular Sciences, KU Leuven), goat anti-SEC23 (Santa Cruz, cat. no. sc-12107; IF 1:100), rabbit anti-SEC23IP (Sigma, cat. no. HPA038403; IF 1:100), rabbit and mouse anti-ERGIC53 (Santa Cruz, SC66880 and SC271517; IF 1:100), mouse anti-multi ubiquitin (Cayman Chemicals, cat. no. 14220; IF 1:100), mouse anti-GAPDH (Biogenesis, cat. no. 4699-955; WB 1:10000), rabbit anti-LC3 (Sigma, cat. no L7543; WB 1:2000), rabbit anti-ARCN1 (Sigma, cat. no. HPA037573; WB 1:1000), rabbit anti-COPB2 (Novus Biologicals, cat. no. NB120-2899; WB 1:1000) mouse anti-beta COP (a gift from the late Thomas Kreiss), hamster anti-ATG9 (a gift of S. Tooze, The Francis Crick Institute, London), rabbit anti-ATG9 (Cell Signalling, cat. no. 13509; IF 1:100), rabbit anti-ATG9 (GeneTex, cat. no. GTX128427; IF 1:100), goat anti-mouse fluorescein isothiocyanate (FITC) (Jackson ImmunoResearch, cat. no. 115-095-062; 1:100), goat anti-rabbit FITC (Jackson ImmunoResearch, cat. no. 111-085-045; 1:100), goat anti-mouse tetramethylrhodamine TRITC (Jackson ImmunoResearch, cat. no. 115-025-062; 1:100), goat anti-rabbit TRITC (Jackson ImmunoResearch, cat. no. 115-025-144; 1:100), goat anti-mouse Alexa Fluor 647 (Molecular Probes, A-21236; 1:500-1:1000), goat anti-rabbit Alexa Fluor 647 (Molecular Probes, A-21244; 1:500-1:1000), CF568 goat anti-mouse IgG (Biotium, cat. no. 20301; 1:100) and CF568 goat anti-rabbit IgG (Biotium, cat. no. 20099; 1:100).

    Techniques: Microscopy, Live Cell Imaging, Expressing, Stable Transfection, Confocal Laser Scanning Microscopy

    Correlative light and electron microscopy of ATG13 and ER. HEK293 cells stably expressing GFP-ATG13 and transiently expressing mCherry-dgk1 (ER marker) were starved, subjected to live-cell imaging by wide-field microscopy and fixed on stage. ( a ) Fluorescent images of the frame capture just before the fixation, × 100 and × 10 DIC images of the fixed cells are shown. Red box in × 10 DIC image indicates the cell of interest. ( b ) Image of the resin-embedded sample. Cell of interest located in red box. ( c ) Resin blocks were trimmed down to a block face of 1 mm 2 and mounted on stub for imaging in an Auriga focused ion beam scanning electron microscopy (FIB-SEM, Carl Zeiss). Overview images before (left) and after milling (right) indicating the cell of interest with a red box. ( d ) Montage of an ATG13 particle formation from the live-cell imaging step and z stacks after fixation (particle ii in f ). ( e ) Overlays of light and electron microscopy images. Light and electron microscopy images were correlated using landmarks identified in both (shown in white and green lines, circles and triangles). ( f ) Three-dimensional (3D) opacity rendering of the FIB-SEM image stack. The areas outlined in red within the green boxes indicate ATG13 particles. Particle ii is the one that could be traced throughout the experiment and was identified in both live-cell and FIB-SEM imaging. ATG13 Particles in boxes i and iii could be identified from the wide-field and fluorescence image, but their provenance by live imaging could not because they were on a different focal plane from particle ii. ( g ) Magnification of the area within the green boxes in f (i–iii). Shown are the XY view from the middle of the ATG13 signal, and orthogonal XZ and YZ views along the thin white lines. ( h ) 3D Opacity rendering of the cropped FIB-SEM stacks in g with overlay of the ATG13 signal (red). Rendered in green are the membranes detected in the FIB-SEM stack that are in proximity of the ATG13 particle. Stars indicate mitochondrial membranes. Bars: 10 μm ( a ), 50 μm ( b ), 5 μm ( d – e ), 1 μm ( f ) and 0.25 μm ( g ).

    Journal: Nature Communications

    Article Title: Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles

    doi: 10.1038/ncomms12420

    Figure Lengend Snippet: Correlative light and electron microscopy of ATG13 and ER. HEK293 cells stably expressing GFP-ATG13 and transiently expressing mCherry-dgk1 (ER marker) were starved, subjected to live-cell imaging by wide-field microscopy and fixed on stage. ( a ) Fluorescent images of the frame capture just before the fixation, × 100 and × 10 DIC images of the fixed cells are shown. Red box in × 10 DIC image indicates the cell of interest. ( b ) Image of the resin-embedded sample. Cell of interest located in red box. ( c ) Resin blocks were trimmed down to a block face of 1 mm 2 and mounted on stub for imaging in an Auriga focused ion beam scanning electron microscopy (FIB-SEM, Carl Zeiss). Overview images before (left) and after milling (right) indicating the cell of interest with a red box. ( d ) Montage of an ATG13 particle formation from the live-cell imaging step and z stacks after fixation (particle ii in f ). ( e ) Overlays of light and electron microscopy images. Light and electron microscopy images were correlated using landmarks identified in both (shown in white and green lines, circles and triangles). ( f ) Three-dimensional (3D) opacity rendering of the FIB-SEM image stack. The areas outlined in red within the green boxes indicate ATG13 particles. Particle ii is the one that could be traced throughout the experiment and was identified in both live-cell and FIB-SEM imaging. ATG13 Particles in boxes i and iii could be identified from the wide-field and fluorescence image, but their provenance by live imaging could not because they were on a different focal plane from particle ii. ( g ) Magnification of the area within the green boxes in f (i–iii). Shown are the XY view from the middle of the ATG13 signal, and orthogonal XZ and YZ views along the thin white lines. ( h ) 3D Opacity rendering of the cropped FIB-SEM stacks in g with overlay of the ATG13 signal (red). Rendered in green are the membranes detected in the FIB-SEM stack that are in proximity of the ATG13 particle. Stars indicate mitochondrial membranes. Bars: 10 μm ( a ), 50 μm ( b ), 5 μm ( d – e ), 1 μm ( f ) and 0.25 μm ( g ).

    Article Snippet: The antibodies used in the course of this work were: mouse anti-ATG13 (Millipore, cat. no. MABC46; immuno-fluorescence (IF) 1:100), rabbit anti-ATG101 (Sigma, cat. no. SAB4200175; IF 1:100), mouse anti-WIPI2 (AbD Serotec, cat. no. MCA5780GA; IF 1:200), rabbit anti-ATG16 (MBL, cat. no. PM040; IF 1:100), mouse anti-GFP (Roche, cat. no. 11814460001; IF 1:100), rabbit anti-GFP (gift of L. Roderick, Department of Cardiovascular Sciences, KU Leuven), goat anti-SEC23 (Santa Cruz, cat. no. sc-12107; IF 1:100), rabbit anti-SEC23IP (Sigma, cat. no. HPA038403; IF 1:100), rabbit and mouse anti-ERGIC53 (Santa Cruz, SC66880 and SC271517; IF 1:100), mouse anti-multi ubiquitin (Cayman Chemicals, cat. no. 14220; IF 1:100), mouse anti-GAPDH (Biogenesis, cat. no. 4699-955; WB 1:10000), rabbit anti-LC3 (Sigma, cat. no L7543; WB 1:2000), rabbit anti-ARCN1 (Sigma, cat. no. HPA037573; WB 1:1000), rabbit anti-COPB2 (Novus Biologicals, cat. no. NB120-2899; WB 1:1000) mouse anti-beta COP (a gift from the late Thomas Kreiss), hamster anti-ATG9 (a gift of S. Tooze, The Francis Crick Institute, London), rabbit anti-ATG9 (Cell Signalling, cat. no. 13509; IF 1:100), rabbit anti-ATG9 (GeneTex, cat. no. GTX128427; IF 1:100), goat anti-mouse fluorescein isothiocyanate (FITC) (Jackson ImmunoResearch, cat. no. 115-095-062; 1:100), goat anti-rabbit FITC (Jackson ImmunoResearch, cat. no. 111-085-045; 1:100), goat anti-mouse tetramethylrhodamine TRITC (Jackson ImmunoResearch, cat. no. 115-025-062; 1:100), goat anti-rabbit TRITC (Jackson ImmunoResearch, cat. no. 115-025-144; 1:100), goat anti-mouse Alexa Fluor 647 (Molecular Probes, A-21236; 1:500-1:1000), goat anti-rabbit Alexa Fluor 647 (Molecular Probes, A-21244; 1:500-1:1000), CF568 goat anti-mouse IgG (Biotium, cat. no. 20301; 1:100) and CF568 goat anti-rabbit IgG (Biotium, cat. no. 20099; 1:100).

    Techniques: Electron Microscopy, Stable Transfection, Expressing, Marker, Live Cell Imaging, Microscopy, Blocking Assay, Imaging, Fluorescence

    Autophagosomes associate with ATG9 and ERGIC membrane compartments. ( a , b ) HEK293 cells were starved in the presence or absence of VPS34 inhibitor for 1 h, immunolabelled for ATG13 and ATG9 ( a ) or FIP200 and ERGIC53 ( b ), and imaged by dSTORM. ( c , d ) HEK293 cells stably expressing GFP-ATG13 were starved in the presence or absence of Vps34 inhibitor for 1 h, immunolabelled for ATG13 and ATG9 ( c ) or SEC23 ( d ), and imaged by dSTORM. Conventional images and super-resolution magnifications are shown. Scale bars in wide-field images: 5 μm. Scale bars in super-resolution images, 0.5 μm.

    Journal: Nature Communications

    Article Title: Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles

    doi: 10.1038/ncomms12420

    Figure Lengend Snippet: Autophagosomes associate with ATG9 and ERGIC membrane compartments. ( a , b ) HEK293 cells were starved in the presence or absence of VPS34 inhibitor for 1 h, immunolabelled for ATG13 and ATG9 ( a ) or FIP200 and ERGIC53 ( b ), and imaged by dSTORM. ( c , d ) HEK293 cells stably expressing GFP-ATG13 were starved in the presence or absence of Vps34 inhibitor for 1 h, immunolabelled for ATG13 and ATG9 ( c ) or SEC23 ( d ), and imaged by dSTORM. Conventional images and super-resolution magnifications are shown. Scale bars in wide-field images: 5 μm. Scale bars in super-resolution images, 0.5 μm.

    Article Snippet: The antibodies used in the course of this work were: mouse anti-ATG13 (Millipore, cat. no. MABC46; immuno-fluorescence (IF) 1:100), rabbit anti-ATG101 (Sigma, cat. no. SAB4200175; IF 1:100), mouse anti-WIPI2 (AbD Serotec, cat. no. MCA5780GA; IF 1:200), rabbit anti-ATG16 (MBL, cat. no. PM040; IF 1:100), mouse anti-GFP (Roche, cat. no. 11814460001; IF 1:100), rabbit anti-GFP (gift of L. Roderick, Department of Cardiovascular Sciences, KU Leuven), goat anti-SEC23 (Santa Cruz, cat. no. sc-12107; IF 1:100), rabbit anti-SEC23IP (Sigma, cat. no. HPA038403; IF 1:100), rabbit and mouse anti-ERGIC53 (Santa Cruz, SC66880 and SC271517; IF 1:100), mouse anti-multi ubiquitin (Cayman Chemicals, cat. no. 14220; IF 1:100), mouse anti-GAPDH (Biogenesis, cat. no. 4699-955; WB 1:10000), rabbit anti-LC3 (Sigma, cat. no L7543; WB 1:2000), rabbit anti-ARCN1 (Sigma, cat. no. HPA037573; WB 1:1000), rabbit anti-COPB2 (Novus Biologicals, cat. no. NB120-2899; WB 1:1000) mouse anti-beta COP (a gift from the late Thomas Kreiss), hamster anti-ATG9 (a gift of S. Tooze, The Francis Crick Institute, London), rabbit anti-ATG9 (Cell Signalling, cat. no. 13509; IF 1:100), rabbit anti-ATG9 (GeneTex, cat. no. GTX128427; IF 1:100), goat anti-mouse fluorescein isothiocyanate (FITC) (Jackson ImmunoResearch, cat. no. 115-095-062; 1:100), goat anti-rabbit FITC (Jackson ImmunoResearch, cat. no. 111-085-045; 1:100), goat anti-mouse tetramethylrhodamine TRITC (Jackson ImmunoResearch, cat. no. 115-025-062; 1:100), goat anti-rabbit TRITC (Jackson ImmunoResearch, cat. no. 115-025-144; 1:100), goat anti-mouse Alexa Fluor 647 (Molecular Probes, A-21236; 1:500-1:1000), goat anti-rabbit Alexa Fluor 647 (Molecular Probes, A-21244; 1:500-1:1000), CF568 goat anti-mouse IgG (Biotium, cat. no. 20301; 1:100) and CF568 goat anti-rabbit IgG (Biotium, cat. no. 20099; 1:100).

    Techniques: Stable Transfection, Expressing

    Impact of siRNA knockdown on the localization of syntaxin 6 or VAMP4 to the chlamydial inclusion. HeLa cells were treated with control (nontargeting), syntaxin 6, or VAMP4 siRNA and infected with C. trachomatis ( Ct ) for 18 h. Then, cells were fixed and

    Journal:

    Article Title: Vesicle-Associated Membrane Protein 4 and Syntaxin 6 Interactions at the Chlamydial Inclusion

    doi: 10.1128/IAI.00584-13

    Figure Lengend Snippet: Impact of siRNA knockdown on the localization of syntaxin 6 or VAMP4 to the chlamydial inclusion. HeLa cells were treated with control (nontargeting), syntaxin 6, or VAMP4 siRNA and infected with C. trachomatis ( Ct ) for 18 h. Then, cells were fixed and

    Article Snippet: Samples were analyzed by Western blotting with the primary antibodies mouse anti-M2 FLAG (Sigma) (to detect 3×FLAG-syntaxin 6) and rabbit anti-VAMP4 (Sigma) and secondary antibodies goat anti-rabbit IRDye 800CW- and goat anti-mouse IRDye 680LT-conjugated antibodies (LiCor Biosciences, Lincoln, NE).

    Techniques: Infection

    Thin-layer chromatography analysis of VAMP4 and syntaxin 6 in NBD-sphingomyelin trafficking to the chlamydial inclusion. Nontargeting (NT; control), VAMP4 (V4), or syntaxin 6 (Stx6) siRNA-treated cells were infected with C. trachomatis , labeled with fluorescent

    Journal:

    Article Title: Vesicle-Associated Membrane Protein 4 and Syntaxin 6 Interactions at the Chlamydial Inclusion

    doi: 10.1128/IAI.00584-13

    Figure Lengend Snippet: Thin-layer chromatography analysis of VAMP4 and syntaxin 6 in NBD-sphingomyelin trafficking to the chlamydial inclusion. Nontargeting (NT; control), VAMP4 (V4), or syntaxin 6 (Stx6) siRNA-treated cells were infected with C. trachomatis , labeled with fluorescent

    Article Snippet: Samples were analyzed by Western blotting with the primary antibodies mouse anti-M2 FLAG (Sigma) (to detect 3×FLAG-syntaxin 6) and rabbit anti-VAMP4 (Sigma) and secondary antibodies goat anti-rabbit IRDye 800CW- and goat anti-mouse IRDye 680LT-conjugated antibodies (LiCor Biosciences, Lincoln, NE).

    Techniques: Thin Layer Chromatography, Infection, Labeling

    Characterization of syntaxin 6 and VAMP4 interactions in Chlamydia -infected cells. (A) Coimmunoprecipitation of syntaxin 6 and VAMP4 in Chlamydia -infected cells. C2BBe1 cells were transfected with 3×FLAG-syntaxin 6 (3×FL-Stx6) or 3×FLAG

    Journal:

    Article Title: Vesicle-Associated Membrane Protein 4 and Syntaxin 6 Interactions at the Chlamydial Inclusion

    doi: 10.1128/IAI.00584-13

    Figure Lengend Snippet: Characterization of syntaxin 6 and VAMP4 interactions in Chlamydia -infected cells. (A) Coimmunoprecipitation of syntaxin 6 and VAMP4 in Chlamydia -infected cells. C2BBe1 cells were transfected with 3×FLAG-syntaxin 6 (3×FL-Stx6) or 3×FLAG

    Article Snippet: Samples were analyzed by Western blotting with the primary antibodies mouse anti-M2 FLAG (Sigma) (to detect 3×FLAG-syntaxin 6) and rabbit anti-VAMP4 (Sigma) and secondary antibodies goat anti-rabbit IRDye 800CW- and goat anti-mouse IRDye 680LT-conjugated antibodies (LiCor Biosciences, Lincoln, NE).

    Techniques: Infection, Transfection

    Live-cell imaging of NBD-lipid trafficking to the chlamydial inclusions of multiple species in VAMP4 knockdown cells. Cells were transfected with siRNA and infected with different chlamydial species, labeled with fluorescent C 6 -NBD-ceramide (NBD-lipid),

    Journal:

    Article Title: Vesicle-Associated Membrane Protein 4 and Syntaxin 6 Interactions at the Chlamydial Inclusion

    doi: 10.1128/IAI.00584-13

    Figure Lengend Snippet: Live-cell imaging of NBD-lipid trafficking to the chlamydial inclusions of multiple species in VAMP4 knockdown cells. Cells were transfected with siRNA and infected with different chlamydial species, labeled with fluorescent C 6 -NBD-ceramide (NBD-lipid),

    Article Snippet: Samples were analyzed by Western blotting with the primary antibodies mouse anti-M2 FLAG (Sigma) (to detect 3×FLAG-syntaxin 6) and rabbit anti-VAMP4 (Sigma) and secondary antibodies goat anti-rabbit IRDye 800CW- and goat anti-mouse IRDye 680LT-conjugated antibodies (LiCor Biosciences, Lincoln, NE).

    Techniques: Live Cell Imaging, Transfection, Infection, Labeling

    Examination of syntaxin 6 and VAMP4 colocalization at the chlamydial inclusion. HeLa cells were infected with C. trachomatis ( Ct ) for 16 h and then either mock treated or treated with brefeldin A for an additional 2 h. Then, cells were fixed and processed

    Journal:

    Article Title: Vesicle-Associated Membrane Protein 4 and Syntaxin 6 Interactions at the Chlamydial Inclusion

    doi: 10.1128/IAI.00584-13

    Figure Lengend Snippet: Examination of syntaxin 6 and VAMP4 colocalization at the chlamydial inclusion. HeLa cells were infected with C. trachomatis ( Ct ) for 16 h and then either mock treated or treated with brefeldin A for an additional 2 h. Then, cells were fixed and processed

    Article Snippet: Samples were analyzed by Western blotting with the primary antibodies mouse anti-M2 FLAG (Sigma) (to detect 3×FLAG-syntaxin 6) and rabbit anti-VAMP4 (Sigma) and secondary antibodies goat anti-rabbit IRDye 800CW- and goat anti-mouse IRDye 680LT-conjugated antibodies (LiCor Biosciences, Lincoln, NE).

    Techniques: Infection

    3×FLAG-VAMP4 localization to different chlamydial species. HeLa cells were transfected with 3×FLAG-VAMP4 (A) or 3×FLAG vector only (B) and infected with C. trachomatis serovar L2 ( Ct L2) or D ( Ct D), C. muridarum (MoPn [mouse pneumonitis

    Journal:

    Article Title: Vesicle-Associated Membrane Protein 4 and Syntaxin 6 Interactions at the Chlamydial Inclusion

    doi: 10.1128/IAI.00584-13

    Figure Lengend Snippet: 3×FLAG-VAMP4 localization to different chlamydial species. HeLa cells were transfected with 3×FLAG-VAMP4 (A) or 3×FLAG vector only (B) and infected with C. trachomatis serovar L2 ( Ct L2) or D ( Ct D), C. muridarum (MoPn [mouse pneumonitis

    Article Snippet: Samples were analyzed by Western blotting with the primary antibodies mouse anti-M2 FLAG (Sigma) (to detect 3×FLAG-syntaxin 6) and rabbit anti-VAMP4 (Sigma) and secondary antibodies goat anti-rabbit IRDye 800CW- and goat anti-mouse IRDye 680LT-conjugated antibodies (LiCor Biosciences, Lincoln, NE).

    Techniques: Transfection, Plasmid Preparation, Infection

    VAMP4 localization to chlamydial inclusion in the absence of chlamydial protein synthesis. HeLa cells were seeded onto glass coverslips in 24-well plates 24 h prior to infection with C. trachomatis serovar L2 ( Ct ). After 16 h, cells were either fixed

    Journal:

    Article Title: Vesicle-Associated Membrane Protein 4 and Syntaxin 6 Interactions at the Chlamydial Inclusion

    doi: 10.1128/IAI.00584-13

    Figure Lengend Snippet: VAMP4 localization to chlamydial inclusion in the absence of chlamydial protein synthesis. HeLa cells were seeded onto glass coverslips in 24-well plates 24 h prior to infection with C. trachomatis serovar L2 ( Ct ). After 16 h, cells were either fixed

    Article Snippet: Samples were analyzed by Western blotting with the primary antibodies mouse anti-M2 FLAG (Sigma) (to detect 3×FLAG-syntaxin 6) and rabbit anti-VAMP4 (Sigma) and secondary antibodies goat anti-rabbit IRDye 800CW- and goat anti-mouse IRDye 680LT-conjugated antibodies (LiCor Biosciences, Lincoln, NE).

    Techniques: Infection

    Live-cell imaging of NBD-lipid trafficking to the chlamydial inclusion in VAMP4 and syntaxin 6 knockdown cells. Nontargeting (NT; control), VAMP4, or syntaxin 6 (Stx6) siRNA-treated cells were infected with C. trachomatis serovar L2 for 20 h. Cells were

    Journal:

    Article Title: Vesicle-Associated Membrane Protein 4 and Syntaxin 6 Interactions at the Chlamydial Inclusion

    doi: 10.1128/IAI.00584-13

    Figure Lengend Snippet: Live-cell imaging of NBD-lipid trafficking to the chlamydial inclusion in VAMP4 and syntaxin 6 knockdown cells. Nontargeting (NT; control), VAMP4, or syntaxin 6 (Stx6) siRNA-treated cells were infected with C. trachomatis serovar L2 for 20 h. Cells were

    Article Snippet: Samples were analyzed by Western blotting with the primary antibodies mouse anti-M2 FLAG (Sigma) (to detect 3×FLAG-syntaxin 6) and rabbit anti-VAMP4 (Sigma) and secondary antibodies goat anti-rabbit IRDye 800CW- and goat anti-mouse IRDye 680LT-conjugated antibodies (LiCor Biosciences, Lincoln, NE).

    Techniques: Live Cell Imaging, Infection

    Effect of syntaxin 6 and VAMP4 on chlamydial infectious progeny. HeLa cells (VAMP4) or C2BBe1 cells (syntaxin 6) were seeded in 24-well plates and treated with the indicated siRNA. Infectious progeny were determined essentially as described in Materials

    Journal:

    Article Title: Vesicle-Associated Membrane Protein 4 and Syntaxin 6 Interactions at the Chlamydial Inclusion

    doi: 10.1128/IAI.00584-13

    Figure Lengend Snippet: Effect of syntaxin 6 and VAMP4 on chlamydial infectious progeny. HeLa cells (VAMP4) or C2BBe1 cells (syntaxin 6) were seeded in 24-well plates and treated with the indicated siRNA. Infectious progeny were determined essentially as described in Materials

    Article Snippet: Samples were analyzed by Western blotting with the primary antibodies mouse anti-M2 FLAG (Sigma) (to detect 3×FLAG-syntaxin 6) and rabbit anti-VAMP4 (Sigma) and secondary antibodies goat anti-rabbit IRDye 800CW- and goat anti-mouse IRDye 680LT-conjugated antibodies (LiCor Biosciences, Lincoln, NE).

    Techniques:

    CD4 T cells home to the choroid plexus (CP) in an activation-, chemokine-, and intercellular adhesion molecule 1 (ICAM-1)-dependant manner. Intact lateral ventricle (LV) CPs from non-perfused CD45.2 mice, with or without lipopolysaccharide (LPS) preconditioning, were cocultured ex vivo with CD45.1 CD4 T cells in artificial CSF (aCSF) and then analyzed with flow cytometry and confocal microscopy. (A–E) Ex vivo cocultures, showing the homing of activated T cells to the CP with flow cytometry and live-cell imaging, and their impact on gene expression. (A) Flow cytometry analysis of the FSC-SSC T-cell population in untreated (UT) CPs cocultured for 24 h with CD45.1 + carboxyfluorescein succinimidyl ester-labeled non-activated (Non-activated CD4 + ; n = 4) or activated (Activated Th1; n = 4) T cells. (B,C) Confocal live-cell imaging of UT, GFP-labeled CPs, which were derived from UBC-GFP mice (B) and cocultured with SNARF-1 + Th1 cells adhering to and migrating within the CP [ (C) ; yellow arrows indicate interactions]. Scale bars represent 500 µm (B) , 20 µm (C) . (D) Homing kinetics of activated Th1 cells to the CP, after 1, 2, 5, and 24 h ( n = 5 at each time point) of ex vivo coculturing with untreated CPs. (E) A heat-map representation (fold change from control aCSF) of a quantitative PCR analysis of genes encoding immune mediators, performed on total RNA isolated from LV CPs that were cocultured with either non-activated CD4 + ( n = 4) or activated ( n = 4) Th1 cells, as compared with an aCSF control ( n = 3), 4 h after initiating the coculture. Fold changes and P values are provided in Tables S3A,B in Supplementary Material. (F,G) The role of chemokines and cell adhesion molecules in T-cell homing to the CP. (F) LV CPs were isolated from mice with or without LPS preconditioning, and they were then cocultured for 24 h with CD45.1 activated Th1 cells, either with a prior pre-treatment of the chemokine-signaling inhibitor pertussis toxin (PTX) ( n = 5 for LPS-preconditioned and n = 5 for phosphate-buffered saline-injected mice, respectively), or without it ( n = 5 and n = 4, respectively). Then, the CPs were analyzed by flow cytometry. The graph shows the frequency of CP-homing T cells. (G) CPs were isolated from LPS-preconditioned mice and then cultured for 24 h with CD45.1 activated Th1 cells in the presence of ICAM-1 neutralizing antibodies, or an isotype control ( n = 5 in each group). The graph shows the frequency of CP-homing T cells. Each symbol represents one LV CP from an individual mouse. Bars represent means ± SEM. * P

    Journal: Frontiers in Immunology

    Article Title: The Choroid Plexus Functions as a Niche for T-Cell Stimulation Within the Central Nervous System

    doi: 10.3389/fimmu.2018.01066

    Figure Lengend Snippet: CD4 T cells home to the choroid plexus (CP) in an activation-, chemokine-, and intercellular adhesion molecule 1 (ICAM-1)-dependant manner. Intact lateral ventricle (LV) CPs from non-perfused CD45.2 mice, with or without lipopolysaccharide (LPS) preconditioning, were cocultured ex vivo with CD45.1 CD4 T cells in artificial CSF (aCSF) and then analyzed with flow cytometry and confocal microscopy. (A–E) Ex vivo cocultures, showing the homing of activated T cells to the CP with flow cytometry and live-cell imaging, and their impact on gene expression. (A) Flow cytometry analysis of the FSC-SSC T-cell population in untreated (UT) CPs cocultured for 24 h with CD45.1 + carboxyfluorescein succinimidyl ester-labeled non-activated (Non-activated CD4 + ; n = 4) or activated (Activated Th1; n = 4) T cells. (B,C) Confocal live-cell imaging of UT, GFP-labeled CPs, which were derived from UBC-GFP mice (B) and cocultured with SNARF-1 + Th1 cells adhering to and migrating within the CP [ (C) ; yellow arrows indicate interactions]. Scale bars represent 500 µm (B) , 20 µm (C) . (D) Homing kinetics of activated Th1 cells to the CP, after 1, 2, 5, and 24 h ( n = 5 at each time point) of ex vivo coculturing with untreated CPs. (E) A heat-map representation (fold change from control aCSF) of a quantitative PCR analysis of genes encoding immune mediators, performed on total RNA isolated from LV CPs that were cocultured with either non-activated CD4 + ( n = 4) or activated ( n = 4) Th1 cells, as compared with an aCSF control ( n = 3), 4 h after initiating the coculture. Fold changes and P values are provided in Tables S3A,B in Supplementary Material. (F,G) The role of chemokines and cell adhesion molecules in T-cell homing to the CP. (F) LV CPs were isolated from mice with or without LPS preconditioning, and they were then cocultured for 24 h with CD45.1 activated Th1 cells, either with a prior pre-treatment of the chemokine-signaling inhibitor pertussis toxin (PTX) ( n = 5 for LPS-preconditioned and n = 5 for phosphate-buffered saline-injected mice, respectively), or without it ( n = 5 and n = 4, respectively). Then, the CPs were analyzed by flow cytometry. The graph shows the frequency of CP-homing T cells. (G) CPs were isolated from LPS-preconditioned mice and then cultured for 24 h with CD45.1 activated Th1 cells in the presence of ICAM-1 neutralizing antibodies, or an isotype control ( n = 5 in each group). The graph shows the frequency of CP-homing T cells. Each symbol represents one LV CP from an individual mouse. Bars represent means ± SEM. * P

    Article Snippet: Rabbit anti-Claudin-1 (CLD-1, 1:100) was purchased from Proteintech (Chicago, IL, USA); Armenian hamster anti-CD54 (ICAM-1, 1:100) was purchased from BD biosciences (Franklin Lane, NJ, USA); rabbit anti-Iba-1 (1:1,000) was purchased from WAKO (Osaka, Japan); rat anti-I-A/I-E (MHC II, 1:100) and rat anti-CD4 (1:100) were purchased from BioLegend (San Diego, CA, USA); rabbit anti-Ki-67 (1:250) was purchased from Cell Marque (Rocklin, CA, USA); Armenian hamster anti-CD11c (1:50) was purchased from eBioscience (San Diego, CA, USA); goat anti-CD106 (VCAM-1, 1:100) and goat anti-CD31 (PECAM-1, 1:100) were purchased from R & D systems (Minneapolis, MN, USA).

    Techniques: Activation Assay, Mouse Assay, Ex Vivo, Flow Cytometry, Cytometry, Confocal Microscopy, Live Cell Imaging, Expressing, Labeling, Derivative Assay, Real-time Polymerase Chain Reaction, Isolation, Injection, Cell Culture

    Intracerebroventricularly (ICV) injected activated CD4 T cells adhere to and enter the choroid plexus (CP). CD4 T cells were enriched from CD45.1 + splenocytes and ICV-injected, either non-activated (Non-activated CD4 + ) or following activation in a Th1 polarizing cocktail (Activated Th1), to CD45.2 + mice that were either untreated (UT) or preconditioned for 24 h with an intraperitoneal injection of lipopolysaccharide (LPS) (+LPS). The lateral ventricle (LV) CPs of these mice were analyzed by flow cytometry 1 and 3 days post-injection (dpi) (A,C–I) , by immunohistochemistry (IHC) 3 dpi (B) , and by quantitative PCR (qPCR) 1 dpi (J) . (A) A flow cytometry analysis of gated CD11b − mononuclear cells, showing the frequencies of CD45.1 + CD4 + T cells in UT mice ( n = 4) and in mice that had been ICV-injected with non-activated CD4 T cells ( n = 3) or with activated Th1 cells ( n = 3). (B) Representative IHC images, showing the LV CPs in brain sections of LPS-preconditioned mice, 3 dpi of activated Th1 cells immunolabeled with anti-CD4 (green), anti-intercellular adhesion molecule 1 (ICAM-1) (red), anti-Ki-67 (blue), and a DAPI nucleus counterstain (gray). Yellow and white arrows indicate the adherence of CD4 T cells to and their proliferation foci within the CP, respectively, and between ICAM-1 + epithelial cell borders. Right panels show 3D reconstructions of z-sections (28 µm overall, 0.7 µm/slice) of the framed area. Scale bars represent 200 µm (top left), 50 µm (bottom left), or 10 µm (middle). (C–F) Flow cytometry analyses of LV CPs isolated from mice that had been injected with activated Th1 cells, either without LPS preconditioning (1 dpi, n = 8; 3 dpi, n = 7), with LPS preconditioning (1 dpi, n = 7; 3 dpi, n = 5), and UT mice ( n = 10). (C) Flow cytometry plots of gated CD11b − mononuclear cells. (D) CD45.1 + CD4 + T-cell frequencies in LV CPs, 1 and 3 dpi. (E) CFSE hi frequencies of CD45.1 + CD4 + T cells in LV CPs, 1 and 3 dpi. (F) CD45.1 − CD4 + T-cell frequencies in LV CPs, 1 and 3 dpi. (G) A flow cytometry analysis of FSC hi SSC hi CD11b − ICAM-1 + cells shows the fold change of ICAM-1 median fluorescent intensity (MFI) on CP epithelial cells in mice that had been ICV-injected with activated Th1 cells, either without (1 dpi, n = 5) or with LPS preconditioning (1 dpi, n = 4), and in mice preconditioned with LPS alone (2 dpi, n = 3), compared with mice that had been injected ICV with phosphate-buffered saline (PBS) (1 dpi, n = 5). (H) Fold change of ICAM-1 MFI in CP epithelial cells in mice with ( n = 5) or without ( n = 7) LPS preconditioning and an ICV injection of activated Th1 cells (3 dpi), as compared with UT mice ( n = 10). (I) Correlation between ICAM-1 MFI fold change on CP epithelial cells (compared with untreated mice) and CD45.1 + CD4 + T cells homing to the CPs of UT or LPS-preconditioned mice at 3 dpi ( n = 12). (J) A heat-map representation (fold change from control) of a qPCR analysis of genes encoding immune mediators in LV CPs, which were isolated from mice that had been ICV-injected with PBS (1 dpi, n = 5), mice that had been preconditioned with LPS (2 dpi, n = 3), or mice that had been injected ICV with activated Th1 cells without (1 dpi, n = 5) or with LPS preconditioning (1 dpi, n = 4). Fold changes and P values are provided in Tables S2A,B in Supplementary Material. Each symbol represents one LV CP from an individual mouse. Bars represent means ± SEM. * P

    Journal: Frontiers in Immunology

    Article Title: The Choroid Plexus Functions as a Niche for T-Cell Stimulation Within the Central Nervous System

    doi: 10.3389/fimmu.2018.01066

    Figure Lengend Snippet: Intracerebroventricularly (ICV) injected activated CD4 T cells adhere to and enter the choroid plexus (CP). CD4 T cells were enriched from CD45.1 + splenocytes and ICV-injected, either non-activated (Non-activated CD4 + ) or following activation in a Th1 polarizing cocktail (Activated Th1), to CD45.2 + mice that were either untreated (UT) or preconditioned for 24 h with an intraperitoneal injection of lipopolysaccharide (LPS) (+LPS). The lateral ventricle (LV) CPs of these mice were analyzed by flow cytometry 1 and 3 days post-injection (dpi) (A,C–I) , by immunohistochemistry (IHC) 3 dpi (B) , and by quantitative PCR (qPCR) 1 dpi (J) . (A) A flow cytometry analysis of gated CD11b − mononuclear cells, showing the frequencies of CD45.1 + CD4 + T cells in UT mice ( n = 4) and in mice that had been ICV-injected with non-activated CD4 T cells ( n = 3) or with activated Th1 cells ( n = 3). (B) Representative IHC images, showing the LV CPs in brain sections of LPS-preconditioned mice, 3 dpi of activated Th1 cells immunolabeled with anti-CD4 (green), anti-intercellular adhesion molecule 1 (ICAM-1) (red), anti-Ki-67 (blue), and a DAPI nucleus counterstain (gray). Yellow and white arrows indicate the adherence of CD4 T cells to and their proliferation foci within the CP, respectively, and between ICAM-1 + epithelial cell borders. Right panels show 3D reconstructions of z-sections (28 µm overall, 0.7 µm/slice) of the framed area. Scale bars represent 200 µm (top left), 50 µm (bottom left), or 10 µm (middle). (C–F) Flow cytometry analyses of LV CPs isolated from mice that had been injected with activated Th1 cells, either without LPS preconditioning (1 dpi, n = 8; 3 dpi, n = 7), with LPS preconditioning (1 dpi, n = 7; 3 dpi, n = 5), and UT mice ( n = 10). (C) Flow cytometry plots of gated CD11b − mononuclear cells. (D) CD45.1 + CD4 + T-cell frequencies in LV CPs, 1 and 3 dpi. (E) CFSE hi frequencies of CD45.1 + CD4 + T cells in LV CPs, 1 and 3 dpi. (F) CD45.1 − CD4 + T-cell frequencies in LV CPs, 1 and 3 dpi. (G) A flow cytometry analysis of FSC hi SSC hi CD11b − ICAM-1 + cells shows the fold change of ICAM-1 median fluorescent intensity (MFI) on CP epithelial cells in mice that had been ICV-injected with activated Th1 cells, either without (1 dpi, n = 5) or with LPS preconditioning (1 dpi, n = 4), and in mice preconditioned with LPS alone (2 dpi, n = 3), compared with mice that had been injected ICV with phosphate-buffered saline (PBS) (1 dpi, n = 5). (H) Fold change of ICAM-1 MFI in CP epithelial cells in mice with ( n = 5) or without ( n = 7) LPS preconditioning and an ICV injection of activated Th1 cells (3 dpi), as compared with UT mice ( n = 10). (I) Correlation between ICAM-1 MFI fold change on CP epithelial cells (compared with untreated mice) and CD45.1 + CD4 + T cells homing to the CPs of UT or LPS-preconditioned mice at 3 dpi ( n = 12). (J) A heat-map representation (fold change from control) of a qPCR analysis of genes encoding immune mediators in LV CPs, which were isolated from mice that had been ICV-injected with PBS (1 dpi, n = 5), mice that had been preconditioned with LPS (2 dpi, n = 3), or mice that had been injected ICV with activated Th1 cells without (1 dpi, n = 5) or with LPS preconditioning (1 dpi, n = 4). Fold changes and P values are provided in Tables S2A,B in Supplementary Material. Each symbol represents one LV CP from an individual mouse. Bars represent means ± SEM. * P

    Article Snippet: Rabbit anti-Claudin-1 (CLD-1, 1:100) was purchased from Proteintech (Chicago, IL, USA); Armenian hamster anti-CD54 (ICAM-1, 1:100) was purchased from BD biosciences (Franklin Lane, NJ, USA); rabbit anti-Iba-1 (1:1,000) was purchased from WAKO (Osaka, Japan); rat anti-I-A/I-E (MHC II, 1:100) and rat anti-CD4 (1:100) were purchased from BioLegend (San Diego, CA, USA); rabbit anti-Ki-67 (1:250) was purchased from Cell Marque (Rocklin, CA, USA); Armenian hamster anti-CD11c (1:50) was purchased from eBioscience (San Diego, CA, USA); goat anti-CD106 (VCAM-1, 1:100) and goat anti-CD31 (PECAM-1, 1:100) were purchased from R & D systems (Minneapolis, MN, USA).

    Techniques: Injection, Activation Assay, Mouse Assay, Flow Cytometry, Cytometry, Immunohistochemistry, Real-time Polymerase Chain Reaction, Immunolabeling, Isolation

    CD4 T cells homing to the choroid plexus (CP) locally impact antigen-presenting cell subsets. Activated CD45.1 + Th1 cells were intracerebroventricularly (ICV)-injected into the lateral ventricles (LVs) of either untreated (UT) or lipopolysaccharide (LPS)-preconditioned (+LPS) CD45.2 wild-type mice. The LV CPs of these mice were then analyzed with flow cytometry, either 1 days post-injection (dpi) (A–C) or 3 dpi (A–C,E–H) , and with immunohistochemistry (IHC) 3 dpi (D) . Flow cytometry analyses of gated CD45.2 + CD11b + mononuclear myeloid cells of LV CPs isolated from UT mice ( n = 10) or from mice that had been ICV-injected with Th1 cells, either with (1 dpi, n = 7; 3 dpi, n = 5) or without (1 dpi, n = 8; 3 dpi, n = 7) LPS preconditioning. (A,B) The frequency of CD45.2 + CD11b + mononuclear myeloid cells in individual mice (A) and their expression levels of intercellular adhesion molecule 1 (ICAM-1) (B) . (C) The frequency of CD11c subsets among the myeloid population. (D) Representative IHC images, showing the LV CPs of mice that were preconditioned with LPS and ICV-injected with Th1 cells. Images were taken 3 dpi and immunolabeled with anti-ICAM-1 (green), anti-Ki-67 (red), anti-MHC II (blue), and a DAPI nuclear counterstain (gray). Scale bars: 200 µm (top left), 50 µm (bottom left), and 10 µm (top right). Bottom right panel shows a 3D reconstruction of z-sections (27.3 µm overall, 0.7 µm/slice), of the framed area. (E) The distribution of CX 3 CR1 and MHCII cells among the CD45.2 + CD11b + myeloid subsets of the CP at 3 dpi. (F,G) The frequency of CD11c + cells among CX 3 CR1 + MHCII + cells (top panels) and CX 3 CR1 − MHCII + cells (bottom panels). (H) CD11c median fluorescent intensity (MFI) in CX 3 CR1 + MHCII + and CX 3 CR1 − MHCII + in mice that had been LPS preconditioned and ICV injected with activated Th1 cells. Each symbol represents one LV CP from an individual mouse. Bars represent means ± SEM. * P

    Journal: Frontiers in Immunology

    Article Title: The Choroid Plexus Functions as a Niche for T-Cell Stimulation Within the Central Nervous System

    doi: 10.3389/fimmu.2018.01066

    Figure Lengend Snippet: CD4 T cells homing to the choroid plexus (CP) locally impact antigen-presenting cell subsets. Activated CD45.1 + Th1 cells were intracerebroventricularly (ICV)-injected into the lateral ventricles (LVs) of either untreated (UT) or lipopolysaccharide (LPS)-preconditioned (+LPS) CD45.2 wild-type mice. The LV CPs of these mice were then analyzed with flow cytometry, either 1 days post-injection (dpi) (A–C) or 3 dpi (A–C,E–H) , and with immunohistochemistry (IHC) 3 dpi (D) . Flow cytometry analyses of gated CD45.2 + CD11b + mononuclear myeloid cells of LV CPs isolated from UT mice ( n = 10) or from mice that had been ICV-injected with Th1 cells, either with (1 dpi, n = 7; 3 dpi, n = 5) or without (1 dpi, n = 8; 3 dpi, n = 7) LPS preconditioning. (A,B) The frequency of CD45.2 + CD11b + mononuclear myeloid cells in individual mice (A) and their expression levels of intercellular adhesion molecule 1 (ICAM-1) (B) . (C) The frequency of CD11c subsets among the myeloid population. (D) Representative IHC images, showing the LV CPs of mice that were preconditioned with LPS and ICV-injected with Th1 cells. Images were taken 3 dpi and immunolabeled with anti-ICAM-1 (green), anti-Ki-67 (red), anti-MHC II (blue), and a DAPI nuclear counterstain (gray). Scale bars: 200 µm (top left), 50 µm (bottom left), and 10 µm (top right). Bottom right panel shows a 3D reconstruction of z-sections (27.3 µm overall, 0.7 µm/slice), of the framed area. (E) The distribution of CX 3 CR1 and MHCII cells among the CD45.2 + CD11b + myeloid subsets of the CP at 3 dpi. (F,G) The frequency of CD11c + cells among CX 3 CR1 + MHCII + cells (top panels) and CX 3 CR1 − MHCII + cells (bottom panels). (H) CD11c median fluorescent intensity (MFI) in CX 3 CR1 + MHCII + and CX 3 CR1 − MHCII + in mice that had been LPS preconditioned and ICV injected with activated Th1 cells. Each symbol represents one LV CP from an individual mouse. Bars represent means ± SEM. * P

    Article Snippet: Rabbit anti-Claudin-1 (CLD-1, 1:100) was purchased from Proteintech (Chicago, IL, USA); Armenian hamster anti-CD54 (ICAM-1, 1:100) was purchased from BD biosciences (Franklin Lane, NJ, USA); rabbit anti-Iba-1 (1:1,000) was purchased from WAKO (Osaka, Japan); rat anti-I-A/I-E (MHC II, 1:100) and rat anti-CD4 (1:100) were purchased from BioLegend (San Diego, CA, USA); rabbit anti-Ki-67 (1:250) was purchased from Cell Marque (Rocklin, CA, USA); Armenian hamster anti-CD11c (1:50) was purchased from eBioscience (San Diego, CA, USA); goat anti-CD106 (VCAM-1, 1:100) and goat anti-CD31 (PECAM-1, 1:100) were purchased from R & D systems (Minneapolis, MN, USA).

    Techniques: Injection, Mouse Assay, Flow Cytometry, Cytometry, Immunohistochemistry, Isolation, Expressing, Immunolabeling

    Intracerebroventricularly (ICV)-injected, resting ovalbumin (OVA)-specific CD4 T cells undergo proliferation within the choroid plexus (CP) in a cerebrospinal fluid-antigen-dependent manner. Interferon gamma was injected intra-cisterna magna to wild-type mice, either alone or together with OVA 323–339 or MOG 35–55 (as a control peptide). After 24 h, the mice were ICV-injected with carboxyfluorescein succinimidyl ester (CFSE)-labeled, resting OVA-specific Th1 cells, either without a peptide (Control; n = 5), with OVA 323–339 (OVA; n = 7), or with MOG 35–55 [myelin oligodendrocyte glycoprotein (MOG); n = 4]. At 3-day post-injection, the lateral ventricle (LV) CPs of these mice were analyzed by flow cytometry and immunohistochemistry (IHC). (A) Flow cytometry of isolated CPs. The cellular fraction gated on mononuclear cells shows CD45 + CD4 + and CFSE + T cells. (B–F) IHC images of LV CPs obtained from the control and from the OVA-injected mice. (B) Representative brain sections of OVA-injected and control mice, immunolabeled with anti-intercellular adhesion molecule 1 (ICAM-1) (green). The graph shows fold change in ICAM-1 expression in the LV CPs of OVA-injected mice, normalized to the control mice. Scale bars represent 50 µm. (C) Representative brain sections of OVA-injected and control mice, immunolabeled with anti-CD4 (green), anti-vascular cell adhesion molecule 1 (VCAM-1) (blue), and a DAPI counterstained (gray). Scale bars represent 200 µm (top left and middle left panels), 50 µm (top right and middle right panels), and 10 µm (bottom left panel). The bottom right panel shows a 3D reconstruction of z-sections (25.9 µm overall, 0.7 µm/slice) of the framed area. (D–F) Analyses of the interactions between T cells and myeloid cells, and the proliferation of T cells within the CP. (D) Representative brain sections of OVA-injected mice, immunolabeled with anti-CD4 (green), anti-Iba-1 (red), and a TO-PRO-3 nuclear counterstain (blue). The yellow arrows indicate co-localization of CD4 and Iba-1. The right panel shows a 3D reconstruction of z-sections (9.5 µm overall, 0.5 µm/slice) of the framed area. Scale bars represent 50 µm (left) and 10 µm (middle). (E) Representative brain sections of OVA-injected mice immunolabeled with anti-Ki-67 (red) and anti-CD4 (blue). The yellow arrows indicate proliferating CD4 T cells in the CP. The right panel shows a 3D reconstruction of z-sections (22 µm overall, 2 µm/slice) of the framed area. Scale bars represent 50 µm (left) and 10 µm (right). (F) Expression pattern of CFSE and Ki-67 in CD4 + T cells, which were detected in three 40-µm brain sections of a single LV CP from a mouse injected with OVA and with OVA-specific T cells. (A,B) Each symbol represents one LV CP from an individual mouse. Bars represent means ± SEM. * P

    Journal: Frontiers in Immunology

    Article Title: The Choroid Plexus Functions as a Niche for T-Cell Stimulation Within the Central Nervous System

    doi: 10.3389/fimmu.2018.01066

    Figure Lengend Snippet: Intracerebroventricularly (ICV)-injected, resting ovalbumin (OVA)-specific CD4 T cells undergo proliferation within the choroid plexus (CP) in a cerebrospinal fluid-antigen-dependent manner. Interferon gamma was injected intra-cisterna magna to wild-type mice, either alone or together with OVA 323–339 or MOG 35–55 (as a control peptide). After 24 h, the mice were ICV-injected with carboxyfluorescein succinimidyl ester (CFSE)-labeled, resting OVA-specific Th1 cells, either without a peptide (Control; n = 5), with OVA 323–339 (OVA; n = 7), or with MOG 35–55 [myelin oligodendrocyte glycoprotein (MOG); n = 4]. At 3-day post-injection, the lateral ventricle (LV) CPs of these mice were analyzed by flow cytometry and immunohistochemistry (IHC). (A) Flow cytometry of isolated CPs. The cellular fraction gated on mononuclear cells shows CD45 + CD4 + and CFSE + T cells. (B–F) IHC images of LV CPs obtained from the control and from the OVA-injected mice. (B) Representative brain sections of OVA-injected and control mice, immunolabeled with anti-intercellular adhesion molecule 1 (ICAM-1) (green). The graph shows fold change in ICAM-1 expression in the LV CPs of OVA-injected mice, normalized to the control mice. Scale bars represent 50 µm. (C) Representative brain sections of OVA-injected and control mice, immunolabeled with anti-CD4 (green), anti-vascular cell adhesion molecule 1 (VCAM-1) (blue), and a DAPI counterstained (gray). Scale bars represent 200 µm (top left and middle left panels), 50 µm (top right and middle right panels), and 10 µm (bottom left panel). The bottom right panel shows a 3D reconstruction of z-sections (25.9 µm overall, 0.7 µm/slice) of the framed area. (D–F) Analyses of the interactions between T cells and myeloid cells, and the proliferation of T cells within the CP. (D) Representative brain sections of OVA-injected mice, immunolabeled with anti-CD4 (green), anti-Iba-1 (red), and a TO-PRO-3 nuclear counterstain (blue). The yellow arrows indicate co-localization of CD4 and Iba-1. The right panel shows a 3D reconstruction of z-sections (9.5 µm overall, 0.5 µm/slice) of the framed area. Scale bars represent 50 µm (left) and 10 µm (middle). (E) Representative brain sections of OVA-injected mice immunolabeled with anti-Ki-67 (red) and anti-CD4 (blue). The yellow arrows indicate proliferating CD4 T cells in the CP. The right panel shows a 3D reconstruction of z-sections (22 µm overall, 2 µm/slice) of the framed area. Scale bars represent 50 µm (left) and 10 µm (right). (F) Expression pattern of CFSE and Ki-67 in CD4 + T cells, which were detected in three 40-µm brain sections of a single LV CP from a mouse injected with OVA and with OVA-specific T cells. (A,B) Each symbol represents one LV CP from an individual mouse. Bars represent means ± SEM. * P

    Article Snippet: Rabbit anti-Claudin-1 (CLD-1, 1:100) was purchased from Proteintech (Chicago, IL, USA); Armenian hamster anti-CD54 (ICAM-1, 1:100) was purchased from BD biosciences (Franklin Lane, NJ, USA); rabbit anti-Iba-1 (1:1,000) was purchased from WAKO (Osaka, Japan); rat anti-I-A/I-E (MHC II, 1:100) and rat anti-CD4 (1:100) were purchased from BioLegend (San Diego, CA, USA); rabbit anti-Ki-67 (1:250) was purchased from Cell Marque (Rocklin, CA, USA); Armenian hamster anti-CD11c (1:50) was purchased from eBioscience (San Diego, CA, USA); goat anti-CD106 (VCAM-1, 1:100) and goat anti-CD31 (PECAM-1, 1:100) were purchased from R & D systems (Minneapolis, MN, USA).

    Techniques: Injection, Mouse Assay, Labeling, Flow Cytometry, Cytometry, Immunohistochemistry, Isolation, Immunolabeling, Expressing

    The choroid plexus (CP) as a checkpoint for cell-mediated immunity in the central nervous system (CNS): a suggested model. The CP manufactures most of the cerebrospinal fluid (CSF) and serves as an interface between the blood and the CNS. (A) The CP primarily comprises a fenestrated vasculature, a stroma, and epithelial, whose apical surfaces face the CSF. Inflammatory signals such as IL-1β and tumor necrosis factor activate the CP vasculature and epithelium and induce immune signaling in the CP compartment. (B) As part of this inflammatory reaction, peripheral blood effector and/or memory T cells are recruited to the CP stroma and into the CSF. (C,D) Antigens in the CNS, either self or foreign, which drain into the CSF, are sampled by antigen-presenting cells and presented to CD4 T cells which, thereby, undergo activation and migrate into the CNS parenchyma. (E) Intercellular adhesion molecule 1 and chemokines strongly upregulated at the apical surface of the CP epithelium allow T cells in the CSF to adhere the CP and cross its epithelium back into the CP stroma. (F) Activated CD4 T cells further facilitate cell-mediated immunity in the CNS by preconditioning the CNS for cell migration across the ependymal layer of the ventricle and/or across the parenchymal and meningeal CNS vasculature. T-cell activation in the CP compartment, may not only serve as a checkpoint for cell-mediated immunity in the CNS but also impact the immune network required for brain functioning and repair at steady-state.

    Journal: Frontiers in Immunology

    Article Title: The Choroid Plexus Functions as a Niche for T-Cell Stimulation Within the Central Nervous System

    doi: 10.3389/fimmu.2018.01066

    Figure Lengend Snippet: The choroid plexus (CP) as a checkpoint for cell-mediated immunity in the central nervous system (CNS): a suggested model. The CP manufactures most of the cerebrospinal fluid (CSF) and serves as an interface between the blood and the CNS. (A) The CP primarily comprises a fenestrated vasculature, a stroma, and epithelial, whose apical surfaces face the CSF. Inflammatory signals such as IL-1β and tumor necrosis factor activate the CP vasculature and epithelium and induce immune signaling in the CP compartment. (B) As part of this inflammatory reaction, peripheral blood effector and/or memory T cells are recruited to the CP stroma and into the CSF. (C,D) Antigens in the CNS, either self or foreign, which drain into the CSF, are sampled by antigen-presenting cells and presented to CD4 T cells which, thereby, undergo activation and migrate into the CNS parenchyma. (E) Intercellular adhesion molecule 1 and chemokines strongly upregulated at the apical surface of the CP epithelium allow T cells in the CSF to adhere the CP and cross its epithelium back into the CP stroma. (F) Activated CD4 T cells further facilitate cell-mediated immunity in the CNS by preconditioning the CNS for cell migration across the ependymal layer of the ventricle and/or across the parenchymal and meningeal CNS vasculature. T-cell activation in the CP compartment, may not only serve as a checkpoint for cell-mediated immunity in the CNS but also impact the immune network required for brain functioning and repair at steady-state.

    Article Snippet: Rabbit anti-Claudin-1 (CLD-1, 1:100) was purchased from Proteintech (Chicago, IL, USA); Armenian hamster anti-CD54 (ICAM-1, 1:100) was purchased from BD biosciences (Franklin Lane, NJ, USA); rabbit anti-Iba-1 (1:1,000) was purchased from WAKO (Osaka, Japan); rat anti-I-A/I-E (MHC II, 1:100) and rat anti-CD4 (1:100) were purchased from BioLegend (San Diego, CA, USA); rabbit anti-Ki-67 (1:250) was purchased from Cell Marque (Rocklin, CA, USA); Armenian hamster anti-CD11c (1:50) was purchased from eBioscience (San Diego, CA, USA); goat anti-CD106 (VCAM-1, 1:100) and goat anti-CD31 (PECAM-1, 1:100) were purchased from R & D systems (Minneapolis, MN, USA).

    Techniques: Activation Assay, Migration

    Increased expression of growth factor receptors (VEGFR1, FGFR2,) in lung CSCs. H460 cells and lung CSCs dissociated from spheres were plated into 96-well plates precoated with Collagen IV and cultured 8 h. Then adherent cells were incubated with FITC-conjugated Abs against FGFR2, VEGFR1 and VEGFR2 fixed and stained with Hoechst 33342. Images were acquired using the Cellomics ArrayScan HCS Reader (20X objective) and analyzed using the Target Activation BioApplication Software Module. A, Immunofluorescent images of VEGFR1 and FGFR2 in H460 and CSCs cells (20X objective). B, Fluorescence intensity (pix) of VEGFR1 and FGFR2 plotted against object area. Each point represents a single cell. In figures 8 – 10 red lines show the boundaries of the fluorescence intensity of H460 cells.

    Journal: PLoS ONE

    Article Title: Drug-Selected Human Lung Cancer Stem Cells: Cytokine Network, Tumorigenic and Metastatic Properties

    doi: 10.1371/journal.pone.0003077

    Figure Lengend Snippet: Increased expression of growth factor receptors (VEGFR1, FGFR2,) in lung CSCs. H460 cells and lung CSCs dissociated from spheres were plated into 96-well plates precoated with Collagen IV and cultured 8 h. Then adherent cells were incubated with FITC-conjugated Abs against FGFR2, VEGFR1 and VEGFR2 fixed and stained with Hoechst 33342. Images were acquired using the Cellomics ArrayScan HCS Reader (20X objective) and analyzed using the Target Activation BioApplication Software Module. A, Immunofluorescent images of VEGFR1 and FGFR2 in H460 and CSCs cells (20X objective). B, Fluorescence intensity (pix) of VEGFR1 and FGFR2 plotted against object area. Each point represents a single cell. In figures 8 – 10 red lines show the boundaries of the fluorescence intensity of H460 cells.

    Article Snippet: The Cellomics Array Scan HCS Reader (Cellomics/ ThermoFisher) was used for imaging and analysis of expression of CSCs and embryonic stem cell markers in DSCs.

    Techniques: Expressing, Cell Culture, Incubation, Staining, Activation Assay, Software, Fluorescence

    In vitro differentiation potential of lung cancer sphere cells and drug resistance of CSCs. A, Loss of stem cell marker (CD133) and increase of differentiation markers (CK8/18) by lung CSCs differentiated progenitors. Parental H460 cells and CSCs from tumor spheres were seeded in collagen coated well plates and cultured for 3 weeks in complete RPMI 1640 medium supplemented with 10% FBS. Upper row - cell images in phase –contrast microscopy; in the middle - cells immunofluorescently stained for CD133 and bottom row - cells immunofluorescently stained for CK 8/18. B, Self-renewing ability of differentiated lung cancer cells treated with cisplatin . Relative % of cells generated tumor spheres from single-cell suspension cultures of drug selected CSCs, cells differentiated during 3 weeks and Progenitors of CSCs differentiated for 3 weeks were treated with cisplatin (1 µM) for two days. Surviving cells were transferred into low adherent plates and cultured in semisolid serum free medium supplemented with growth factors. Numbers of formed tumor spheres were determined and presented as percent of control. Control is number of spheres formed by transfer of cells derived from control tumor spheres. Number of these spheres is accepted as 100 %. C, Effect of cisplatin and doxorubicin on proliferation of parental H460 cells, CSCs and their differentiated cells . H460, lung CSCs and differentiated cells were plated in 96-well plates precoated with Collagen at 1×10 4 cells/well in complete RPMI 1640 medium with 10% FBS. After 24 h doxorubicin or cisplatin was added at the indicated concentrations. Cells were cultured for 72 h, fixed, stained with Hoechst 33342 (2 µg/mL), and counted using the Cellomics ArrayScan HCS Reader.

    Journal: PLoS ONE

    Article Title: Drug-Selected Human Lung Cancer Stem Cells: Cytokine Network, Tumorigenic and Metastatic Properties

    doi: 10.1371/journal.pone.0003077

    Figure Lengend Snippet: In vitro differentiation potential of lung cancer sphere cells and drug resistance of CSCs. A, Loss of stem cell marker (CD133) and increase of differentiation markers (CK8/18) by lung CSCs differentiated progenitors. Parental H460 cells and CSCs from tumor spheres were seeded in collagen coated well plates and cultured for 3 weeks in complete RPMI 1640 medium supplemented with 10% FBS. Upper row - cell images in phase –contrast microscopy; in the middle - cells immunofluorescently stained for CD133 and bottom row - cells immunofluorescently stained for CK 8/18. B, Self-renewing ability of differentiated lung cancer cells treated with cisplatin . Relative % of cells generated tumor spheres from single-cell suspension cultures of drug selected CSCs, cells differentiated during 3 weeks and Progenitors of CSCs differentiated for 3 weeks were treated with cisplatin (1 µM) for two days. Surviving cells were transferred into low adherent plates and cultured in semisolid serum free medium supplemented with growth factors. Numbers of formed tumor spheres were determined and presented as percent of control. Control is number of spheres formed by transfer of cells derived from control tumor spheres. Number of these spheres is accepted as 100 %. C, Effect of cisplatin and doxorubicin on proliferation of parental H460 cells, CSCs and their differentiated cells . H460, lung CSCs and differentiated cells were plated in 96-well plates precoated with Collagen at 1×10 4 cells/well in complete RPMI 1640 medium with 10% FBS. After 24 h doxorubicin or cisplatin was added at the indicated concentrations. Cells were cultured for 72 h, fixed, stained with Hoechst 33342 (2 µg/mL), and counted using the Cellomics ArrayScan HCS Reader.

    Article Snippet: The Cellomics Array Scan HCS Reader (Cellomics/ ThermoFisher) was used for imaging and analysis of expression of CSCs and embryonic stem cell markers in DSCs.

    Techniques: In Vitro, Marker, Cell Culture, Microscopy, Staining, Generated, Derivative Assay

    Increased expression of chemokine receptors (CXCR1, 2) in lung CSCs. A, Immunofluorescent images of CXCR1 and CXCR2 in H460 and CS cells. H460 cells and lung CSCs dissociated from spheres were plated into 96-well plates precoated with Collagen IV and cultured 8 h. Then adherent cells were incubated with antibodies against CXCR1 and CXCR2 and with secondary antibodies conjugated with Alexa Fluor® 488 and stained with Hoechst33342. Images were acquired using the Cellomics ArrayScan HCS Reader (20X objective) and analyzed using the Target Activation BioApplication Software Module. B, Fluorescence intensity (pix) of CXCR1 and CXCR2 plotted against object area.

    Journal: PLoS ONE

    Article Title: Drug-Selected Human Lung Cancer Stem Cells: Cytokine Network, Tumorigenic and Metastatic Properties

    doi: 10.1371/journal.pone.0003077

    Figure Lengend Snippet: Increased expression of chemokine receptors (CXCR1, 2) in lung CSCs. A, Immunofluorescent images of CXCR1 and CXCR2 in H460 and CS cells. H460 cells and lung CSCs dissociated from spheres were plated into 96-well plates precoated with Collagen IV and cultured 8 h. Then adherent cells were incubated with antibodies against CXCR1 and CXCR2 and with secondary antibodies conjugated with Alexa Fluor® 488 and stained with Hoechst33342. Images were acquired using the Cellomics ArrayScan HCS Reader (20X objective) and analyzed using the Target Activation BioApplication Software Module. B, Fluorescence intensity (pix) of CXCR1 and CXCR2 plotted against object area.

    Article Snippet: The Cellomics Array Scan HCS Reader (Cellomics/ ThermoFisher) was used for imaging and analysis of expression of CSCs and embryonic stem cell markers in DSCs.

    Techniques: Expressing, Cell Culture, Incubation, Staining, Activation Assay, Software, Fluorescence

    Expression of adhesion molecules, VLA-4(CD49d), VLA-5(CD49e), VLA-6(CD49f), by H460 cells and DSCs. Cells were incubated with Abs against VLA-4-FITC and VLA-6-PC5 or VLA-5-FITC and VLA-6-PC5. Cell images were acquired using the Cellomics ArrayScan HCS Reader (20X, 40X objectives) and analyzed using the Target Activation BioApplication Software Module. A, Immunofluorescent images of VLA4/VLA6 (left) and VLA-5/VLA-6 (right) expression in H460 and DSCs cells (40X objective). B-D, A n average fluorescence intensity of VLA-4(B), VLA-5(C) and VLA-6(D) in H460 cells (black line) and DSCs (grey line).

    Journal: PLoS ONE

    Article Title: Drug-Selected Human Lung Cancer Stem Cells: Cytokine Network, Tumorigenic and Metastatic Properties

    doi: 10.1371/journal.pone.0003077

    Figure Lengend Snippet: Expression of adhesion molecules, VLA-4(CD49d), VLA-5(CD49e), VLA-6(CD49f), by H460 cells and DSCs. Cells were incubated with Abs against VLA-4-FITC and VLA-6-PC5 or VLA-5-FITC and VLA-6-PC5. Cell images were acquired using the Cellomics ArrayScan HCS Reader (20X, 40X objectives) and analyzed using the Target Activation BioApplication Software Module. A, Immunofluorescent images of VLA4/VLA6 (left) and VLA-5/VLA-6 (right) expression in H460 and DSCs cells (40X objective). B-D, A n average fluorescence intensity of VLA-4(B), VLA-5(C) and VLA-6(D) in H460 cells (black line) and DSCs (grey line).

    Article Snippet: The Cellomics Array Scan HCS Reader (Cellomics/ ThermoFisher) was used for imaging and analysis of expression of CSCs and embryonic stem cell markers in DSCs.

    Techniques: Expressing, Incubation, Activation Assay, Software, Fluorescence

    Analysis of β-catenin intracellular distribution in H460 cells and DSCs. Cells were fixed and incubated with Alexa Fluor® 488 phalloidin or with primary Abs against β-catenin and with secondary Alexa Fluor 488 conjugated Abs. Next cells were stained with Hoechst33342. Cell images were acquired using the Cellomics ArrayScan HCS Reader (20X objective) and analyzed using the Compartment Analysis BioApplication Software Module and the Target Activation BioApplication Software Module. A, Images of H460 cells and DSCs immunofluorescently stained for β-catenin (A). B, An average fluorescence intensity of nuclear β-catenin in H460 (black line) and DSCs (grey line) .C, An average fluorescence intensity of cellular phosphor- β-catenin in H460 (black line) and DSCs (grey line). D, Cytoskeleton images of H460 cells and DSCs immunofluorescently stained for phalloidin and Hoechst33342.

    Journal: PLoS ONE

    Article Title: Drug-Selected Human Lung Cancer Stem Cells: Cytokine Network, Tumorigenic and Metastatic Properties

    doi: 10.1371/journal.pone.0003077

    Figure Lengend Snippet: Analysis of β-catenin intracellular distribution in H460 cells and DSCs. Cells were fixed and incubated with Alexa Fluor® 488 phalloidin or with primary Abs against β-catenin and with secondary Alexa Fluor 488 conjugated Abs. Next cells were stained with Hoechst33342. Cell images were acquired using the Cellomics ArrayScan HCS Reader (20X objective) and analyzed using the Compartment Analysis BioApplication Software Module and the Target Activation BioApplication Software Module. A, Images of H460 cells and DSCs immunofluorescently stained for β-catenin (A). B, An average fluorescence intensity of nuclear β-catenin in H460 (black line) and DSCs (grey line) .C, An average fluorescence intensity of cellular phosphor- β-catenin in H460 (black line) and DSCs (grey line). D, Cytoskeleton images of H460 cells and DSCs immunofluorescently stained for phalloidin and Hoechst33342.

    Article Snippet: The Cellomics Array Scan HCS Reader (Cellomics/ ThermoFisher) was used for imaging and analysis of expression of CSCs and embryonic stem cell markers in DSCs.

    Techniques: Incubation, Staining, Software, Activation Assay, Fluorescence

    Expression of growth factor and chemokine receptors in lung CSCs. A, B , H460 cells and lung CSCs dissociated from spheres were plated into 96-well plates precoated with Collagen IV and cultured 8 h. Then adherent cells were immunofluorescently stained for CXCR4 (SDF-1 receptor); images were acquired using the Cellomics ArrayScan HCS Reader (20X objective) and analyzed using the Target Activation BioApplication Software Module. A. Immunofluorescent images of CXCR4 in H460 and CSCs cells. B. Fluorescence intensity (pix) of CXCR4 is plotted against object area. C. Expression of growth factor and chemokine receptors in lung CSCs growing in tumor spheres. Lung tumor spheres were immunofluorescently stained for VEGFR1; FGFR2, CXCR1 and CXCR4 receptors; images were acquired using the Cellomics ArrayScan HCS Reader (10X objective). Immunofluorescent images of lung tumor spheres stained for VEGFR1, FGFR2, CXCR1 and CXCR4 are presented.

    Journal: PLoS ONE

    Article Title: Drug-Selected Human Lung Cancer Stem Cells: Cytokine Network, Tumorigenic and Metastatic Properties

    doi: 10.1371/journal.pone.0003077

    Figure Lengend Snippet: Expression of growth factor and chemokine receptors in lung CSCs. A, B , H460 cells and lung CSCs dissociated from spheres were plated into 96-well plates precoated with Collagen IV and cultured 8 h. Then adherent cells were immunofluorescently stained for CXCR4 (SDF-1 receptor); images were acquired using the Cellomics ArrayScan HCS Reader (20X objective) and analyzed using the Target Activation BioApplication Software Module. A. Immunofluorescent images of CXCR4 in H460 and CSCs cells. B. Fluorescence intensity (pix) of CXCR4 is plotted against object area. C. Expression of growth factor and chemokine receptors in lung CSCs growing in tumor spheres. Lung tumor spheres were immunofluorescently stained for VEGFR1; FGFR2, CXCR1 and CXCR4 receptors; images were acquired using the Cellomics ArrayScan HCS Reader (10X objective). Immunofluorescent images of lung tumor spheres stained for VEGFR1, FGFR2, CXCR1 and CXCR4 are presented.

    Article Snippet: The Cellomics Array Scan HCS Reader (Cellomics/ ThermoFisher) was used for imaging and analysis of expression of CSCs and embryonic stem cell markers in DSCs.

    Techniques: Expressing, Cell Culture, Staining, Activation Assay, Software, Fluorescence

    Analysis of CD133, embryonic stem cell (ESC) markers and cytokeratins 8/18 expression in H460 cells and DSCs. H460 cells and DSCs, growing in 96-well plates, were fixed and incubated with primary Abs against CD133, TRA-1-81, SSEA-3, Oct-4, or cytokeratins8/18 and then with secondary Abs. Cell nuclei were stained with Hoechst 33342. Cell images were acquired using the Cellomics ArrayScan HCS Reader (20X, 40X objectives) and analyzed using the Target Activation BioApplication Software Module. A, Immunofluorescent images of tumor cells. B, Fluorescence intensity (pix) of CD133 plotted against object area. Each point represents a single cell. Cells to the right of the red line are CD133+ (above IgG control staining). C, Images of tumor cells immunofluorescently stained tumor cells for TRA-1-81, SSEA-3 and Oct-4 ES cell markers. D. Fluorescence intensity of TRA-1-81, SSEA-3 and Oct-4, plotted against object area. Each point represents a single cell. Cells to the right of the red line are positive (above IgG control staining). E, Images of immunofluorescently stained tumor cells for cytokeratins8/18. F, Fluorescence intensity of cytokeratins8/18 in H460 cells (black dots) and DSCs (grey dots) plotted against object area .

    Journal: PLoS ONE

    Article Title: Drug-Selected Human Lung Cancer Stem Cells: Cytokine Network, Tumorigenic and Metastatic Properties

    doi: 10.1371/journal.pone.0003077

    Figure Lengend Snippet: Analysis of CD133, embryonic stem cell (ESC) markers and cytokeratins 8/18 expression in H460 cells and DSCs. H460 cells and DSCs, growing in 96-well plates, were fixed and incubated with primary Abs against CD133, TRA-1-81, SSEA-3, Oct-4, or cytokeratins8/18 and then with secondary Abs. Cell nuclei were stained with Hoechst 33342. Cell images were acquired using the Cellomics ArrayScan HCS Reader (20X, 40X objectives) and analyzed using the Target Activation BioApplication Software Module. A, Immunofluorescent images of tumor cells. B, Fluorescence intensity (pix) of CD133 plotted against object area. Each point represents a single cell. Cells to the right of the red line are CD133+ (above IgG control staining). C, Images of tumor cells immunofluorescently stained tumor cells for TRA-1-81, SSEA-3 and Oct-4 ES cell markers. D. Fluorescence intensity of TRA-1-81, SSEA-3 and Oct-4, plotted against object area. Each point represents a single cell. Cells to the right of the red line are positive (above IgG control staining). E, Images of immunofluorescently stained tumor cells for cytokeratins8/18. F, Fluorescence intensity of cytokeratins8/18 in H460 cells (black dots) and DSCs (grey dots) plotted against object area .

    Article Snippet: The Cellomics Array Scan HCS Reader (Cellomics/ ThermoFisher) was used for imaging and analysis of expression of CSCs and embryonic stem cell markers in DSCs.

    Techniques: Expressing, Incubation, Staining, Activation Assay, Software, Fluorescence

    G3BP1 acidic domain increases axonal mRNA translation and disassembles stress granules. a , b Representative images for puromycin (Puro) incorporation in DRG neurons transfected with the indicated constructs are shown ( a ). Significant increase in axonal puromycin signals in the G3BP1 B domain-expressing neurons is seen, with no significant change in the cell body puromycin incorporation ( b ; N ≥ 23 axons over three repetitions; ** p ≤ 0.01, **** p ≤ 0.0001 by one-way ANOVA with Tukey HSD posthoc) [scale bar = 5 μm]. c G3BP1 depleted DRG cultures similarly show increased puromycin incorporation in axons with no significant change in cell body puromycin incorporation ( N ≥ 23 axons over three repetitions; ** p ≤ 0.01, **** p ≤ 0.0001 by one-way ANOVA with Tukey HSD posthoc). See Supplementary Fig. 7a for representative images of these puromycin incorporation studies. d Quantitation of endogenous axonal NRN1, IMPβ1, and GAP43 protein levels in DRG cultures transfected with GFP, G3BP1-GFP, and G3BP1 B domain-GFP is shown. Axonal NRN1 and IMPβ1 but not GAP43 levels are significantly reduced in G3BP1 overexpression. G3BP1 B domain-expressing neurons show significantly higher axonal NRN1, but no change in axonal IMPβ1 and GAP43 levels ( N ≥ 33 axons over three repetitions; * p ≤ 0.05, **** p ≤ 0.0001 by one-way ANOVA with Tukey HSD posthoc). Representative images for axonal immunofluorescence and cell body NRN1, IMPβ1, and GAP43 proteins are shown in Supplementary Fig. 7b, c . e RTddPCR for axonal mRNAs co-precipitating with G3BP1-GFP in DRG neurons are shown as average % mRNA associated with G3BP1-GFP ± SEM. Nrn1 and Impβ1 mRNAs association with G3BP1-GFP significantly reduced by cotransfection with the G3BP1 B domain, but neither RNA coprecipitates with the B domain ( N = 4 culture preparations; * p ≤ 0.05, ** p ≤ 0.01 by Student’s t -test for the indicated data pairs)

    Journal: Nature Communications

    Article Title: Axonal G3BP1 stress granule protein limits axonal mRNA translation and nerve regeneration

    doi: 10.1038/s41467-018-05647-x

    Figure Lengend Snippet: G3BP1 acidic domain increases axonal mRNA translation and disassembles stress granules. a , b Representative images for puromycin (Puro) incorporation in DRG neurons transfected with the indicated constructs are shown ( a ). Significant increase in axonal puromycin signals in the G3BP1 B domain-expressing neurons is seen, with no significant change in the cell body puromycin incorporation ( b ; N ≥ 23 axons over three repetitions; ** p ≤ 0.01, **** p ≤ 0.0001 by one-way ANOVA with Tukey HSD posthoc) [scale bar = 5 μm]. c G3BP1 depleted DRG cultures similarly show increased puromycin incorporation in axons with no significant change in cell body puromycin incorporation ( N ≥ 23 axons over three repetitions; ** p ≤ 0.01, **** p ≤ 0.0001 by one-way ANOVA with Tukey HSD posthoc). See Supplementary Fig. 7a for representative images of these puromycin incorporation studies. d Quantitation of endogenous axonal NRN1, IMPβ1, and GAP43 protein levels in DRG cultures transfected with GFP, G3BP1-GFP, and G3BP1 B domain-GFP is shown. Axonal NRN1 and IMPβ1 but not GAP43 levels are significantly reduced in G3BP1 overexpression. G3BP1 B domain-expressing neurons show significantly higher axonal NRN1, but no change in axonal IMPβ1 and GAP43 levels ( N ≥ 33 axons over three repetitions; * p ≤ 0.05, **** p ≤ 0.0001 by one-way ANOVA with Tukey HSD posthoc). Representative images for axonal immunofluorescence and cell body NRN1, IMPβ1, and GAP43 proteins are shown in Supplementary Fig. 7b, c . e RTddPCR for axonal mRNAs co-precipitating with G3BP1-GFP in DRG neurons are shown as average % mRNA associated with G3BP1-GFP ± SEM. Nrn1 and Impβ1 mRNAs association with G3BP1-GFP significantly reduced by cotransfection with the G3BP1 B domain, but neither RNA coprecipitates with the B domain ( N = 4 culture preparations; * p ≤ 0.05, ** p ≤ 0.01 by Student’s t -test for the indicated data pairs)

    Article Snippet: Primary antibodies consisted of: rabbit anti-G3BP1 (1:200, Sigma), RT97 mouse anti-neurofilament (NF; 1:500, Devel.

    Techniques: Transfection, Construct, Expressing, Quantitation Assay, Over Expression, Immunofluorescence, Cotransfection

    Cell permeable G3BP1 190–208 peptide increases axonal mRNA translation and disassembles stress granules. a , b , Representative images for puromycin incorporation in axons of control, 168–189 peptide and 190–208 peptide-treated DRG cultures are shown ( a ). Quantitation of puromycin incorporation into distal DRG axons under these conditions shows a significant increase in axonal protein synthesis for the 190–208 peptide-treated cultures compared to control and 168–189 peptide exposure ( b ; N ≥ 83 axons over 3 DRG cultures; *** p ≤ 0.005, **** p ≤ 0.0001 by one-way ANOVA with Tukey HSD post-hoc). c FRAP analyses for DRGs for GFP MYR 5 ′ /3 ′ nrn1, GFP MYR 5 ′ /3 ′ impβ1 and GFP MYR 5 ′ /3 ′ gap43 in axons of DRGs expressing BFP or G3BP1-BFP ± 10 µM 190–208 G3BP1 peptide (30 min. treatment). Only translation of GFP MYR 5 ′ /3 ′ nrn1 is increased by the 190–208 peptide with G3BP1 overexpression ( N ≥ 11 axons over three culture repetitions; all statistics were done by one-way ANOVA with Tukey HSD post-hoc: * p ≤ 0.05, ** p ≤ 0.01 for BFP vs. BFP + 190–208 peptide; # p ≤ 0.05 for G3BP1-BFP vs. G3BP1-BFP + 190–208; p ≤ 0.05 for BFP vs. G3BP1; and, † p ≤ 0.05 for BFP vs. G3BP1-BFP + 190–208 peptide; no values for GFP MYR 5 ′ /3 ′ gap43 were statistically significant). d Representative images of G3BP1-mCh in DRG axons under control conditions and after treatment with 190–208 G3BP1 or 168–189 peptides for 15 min. are shown. Axon tracing was generated from DIC images [scale bar = 10 μm]. For representative live cell imaging sequence refer to Supplementary Movie 4 . e Density of G3BP1-mCh aggregates along 100 μm length axons from DRG cultures treated as in d is shown ( N ≥ 38 axons over three repetitions; **** p ≤ 0.0001 by ANOVA with Tukey HSD post-hoc). f Size of G3BP1-mCh aggregate is shown as indicated bins for from DRG cultures treated as in d ( N ≥ 221 aggregates over three repetitions; **** p ≤ 0.0005, ***** p ≤ 0.0001 for entire population distributions by Kolmogorov–Smirnov test)

    Journal: Nature Communications

    Article Title: Axonal G3BP1 stress granule protein limits axonal mRNA translation and nerve regeneration

    doi: 10.1038/s41467-018-05647-x

    Figure Lengend Snippet: Cell permeable G3BP1 190–208 peptide increases axonal mRNA translation and disassembles stress granules. a , b , Representative images for puromycin incorporation in axons of control, 168–189 peptide and 190–208 peptide-treated DRG cultures are shown ( a ). Quantitation of puromycin incorporation into distal DRG axons under these conditions shows a significant increase in axonal protein synthesis for the 190–208 peptide-treated cultures compared to control and 168–189 peptide exposure ( b ; N ≥ 83 axons over 3 DRG cultures; *** p ≤ 0.005, **** p ≤ 0.0001 by one-way ANOVA with Tukey HSD post-hoc). c FRAP analyses for DRGs for GFP MYR 5 ′ /3 ′ nrn1, GFP MYR 5 ′ /3 ′ impβ1 and GFP MYR 5 ′ /3 ′ gap43 in axons of DRGs expressing BFP or G3BP1-BFP ± 10 µM 190–208 G3BP1 peptide (30 min. treatment). Only translation of GFP MYR 5 ′ /3 ′ nrn1 is increased by the 190–208 peptide with G3BP1 overexpression ( N ≥ 11 axons over three culture repetitions; all statistics were done by one-way ANOVA with Tukey HSD post-hoc: * p ≤ 0.05, ** p ≤ 0.01 for BFP vs. BFP + 190–208 peptide; # p ≤ 0.05 for G3BP1-BFP vs. G3BP1-BFP + 190–208; p ≤ 0.05 for BFP vs. G3BP1; and, † p ≤ 0.05 for BFP vs. G3BP1-BFP + 190–208 peptide; no values for GFP MYR 5 ′ /3 ′ gap43 were statistically significant). d Representative images of G3BP1-mCh in DRG axons under control conditions and after treatment with 190–208 G3BP1 or 168–189 peptides for 15 min. are shown. Axon tracing was generated from DIC images [scale bar = 10 μm]. For representative live cell imaging sequence refer to Supplementary Movie 4 . e Density of G3BP1-mCh aggregates along 100 μm length axons from DRG cultures treated as in d is shown ( N ≥ 38 axons over three repetitions; **** p ≤ 0.0001 by ANOVA with Tukey HSD post-hoc). f Size of G3BP1-mCh aggregate is shown as indicated bins for from DRG cultures treated as in d ( N ≥ 221 aggregates over three repetitions; **** p ≤ 0.0005, ***** p ≤ 0.0001 for entire population distributions by Kolmogorov–Smirnov test)

    Article Snippet: Primary antibodies consisted of: rabbit anti-G3BP1 (1:200, Sigma), RT97 mouse anti-neurofilament (NF; 1:500, Devel.

    Techniques: Quantitation Assay, Expressing, Over Expression, Generated, Live Cell Imaging, Sequencing

    G3BP1 localizes to axons in stress granule-like aggregates. a Immunofluorescence for G3BP1 shows signals in the cell body (asterisk) and axons (arrows) of a cultured DRG neuron; arrowheads indicate Schwann cell with prominent G3BP1 immunoreactivity visible in the inset DIC image. Previous work has shown that neurites of these adult DRG neurons have axonal features and lack dendritic features 49 ; we will use ‘axon’ for describing these hereafter [scale bar = 50 µm]. b , c Single planes for axons of naive DRG cultures co-labeled for indicated proteins are shown; box represents the area for high magnification insets to right ( b ). Axonal G3BP1 shows higher colocalization coefficients for SG than PB proteins by Fisher’s Z transformation ( c ; N ≥ 30 axons over 3 repetitions; ** p ≤ 0.01, **** p ≤ 0.001 by one-way ANOVA with Tukey HSD post-hoc) [scale bar = 10 µm for large panels, 1 µm for insets]. d PLA shows higher colocalization for G3BP1 and HuR than G3BP1 and DCP1A (G3BP1 + HuR PLA = 0.038 ± 0.003 and G3BP1 + DCP1A PLA = 0.027 ± 0.002 signals/µm 2 ; N ≥ 40 neurons over 6 repetitions, p = 0.016 by Student’s t -test) [scale bar = 20 µm]. e , f Confocal images for G3BP1 and TIA1 in naive and 7 d post-injured (‘regenerating’) sciatic nerve are shown ( e ). Upper image panels of each pair show G3BP1 and TIA1 merged with NF signals in single plane. Lower panels of each pair show XYZ for G3BP1 and TIA1 signals that overlap with NF across the Z stack Quantitation of axonal G3BP1 and TIA1 signals are shown ( f ) as mean ± SEM ( N = 6 animals; * p ≤ 0.05, *** p ≤ 0.001 for G3BP1 and ## p ≤ 0.01, #### p ≤ 0.0001 for TIA1 by Student’s t -test for vs. naive) [scale bar = 5 µm]. g , h Quantification of G3BP1 levels ( g ) and G3BP1 immunofluorescence ( h ) in axons of DRGs cultured from naive vs. 7 d injury-conditioned animals are shown (mean ± SEM for N ≥ 66 neurons over 3 repetitions; *** p ≤ 0.001 by Student’s t -test) [scale bar 20 µm]

    Journal: Nature Communications

    Article Title: Axonal G3BP1 stress granule protein limits axonal mRNA translation and nerve regeneration

    doi: 10.1038/s41467-018-05647-x

    Figure Lengend Snippet: G3BP1 localizes to axons in stress granule-like aggregates. a Immunofluorescence for G3BP1 shows signals in the cell body (asterisk) and axons (arrows) of a cultured DRG neuron; arrowheads indicate Schwann cell with prominent G3BP1 immunoreactivity visible in the inset DIC image. Previous work has shown that neurites of these adult DRG neurons have axonal features and lack dendritic features 49 ; we will use ‘axon’ for describing these hereafter [scale bar = 50 µm]. b , c Single planes for axons of naive DRG cultures co-labeled for indicated proteins are shown; box represents the area for high magnification insets to right ( b ). Axonal G3BP1 shows higher colocalization coefficients for SG than PB proteins by Fisher’s Z transformation ( c ; N ≥ 30 axons over 3 repetitions; ** p ≤ 0.01, **** p ≤ 0.001 by one-way ANOVA with Tukey HSD post-hoc) [scale bar = 10 µm for large panels, 1 µm for insets]. d PLA shows higher colocalization for G3BP1 and HuR than G3BP1 and DCP1A (G3BP1 + HuR PLA = 0.038 ± 0.003 and G3BP1 + DCP1A PLA = 0.027 ± 0.002 signals/µm 2 ; N ≥ 40 neurons over 6 repetitions, p = 0.016 by Student’s t -test) [scale bar = 20 µm]. e , f Confocal images for G3BP1 and TIA1 in naive and 7 d post-injured (‘regenerating’) sciatic nerve are shown ( e ). Upper image panels of each pair show G3BP1 and TIA1 merged with NF signals in single plane. Lower panels of each pair show XYZ for G3BP1 and TIA1 signals that overlap with NF across the Z stack Quantitation of axonal G3BP1 and TIA1 signals are shown ( f ) as mean ± SEM ( N = 6 animals; * p ≤ 0.05, *** p ≤ 0.001 for G3BP1 and ## p ≤ 0.01, #### p ≤ 0.0001 for TIA1 by Student’s t -test for vs. naive) [scale bar = 5 µm]. g , h Quantification of G3BP1 levels ( g ) and G3BP1 immunofluorescence ( h ) in axons of DRGs cultured from naive vs. 7 d injury-conditioned animals are shown (mean ± SEM for N ≥ 66 neurons over 3 repetitions; *** p ≤ 0.001 by Student’s t -test) [scale bar 20 µm]

    Article Snippet: Primary antibodies consisted of: rabbit anti-G3BP1 (1:200, Sigma), RT97 mouse anti-neurofilament (NF; 1:500, Devel.

    Techniques: Immunofluorescence, Cell Culture, Labeling, Transformation Assay, Proximity Ligation Assay, Quantitation Assay

    G3BP1 is phosphorylated in regenerating axons. a Representative images for axons of DRG neurons transfected with indicated G3BP1 constructs vs. eGFP are shown. G3BP1-GFP and G3BP1 S149A -GFP show prominent aggregates in axons that colocalize with HuR (arrows). In contrast, axonal signals for G3BP1 S149E -GFP and eGFP appear diffuse [scale bar = 5 µm]. b Quantification of axonal aggregates for G3BP1-GFP, G3BP1 S149A -GFP, and G3BP1 S149E -GFP is shown as average ± SEM ( N ≥ 10 neurons over 3 repetitions; *** p ≤ 0.005 by one-way ANOVA with Tukey HSD post-hoc). c FRAP analyses for neurons transfected with constructs as in A are shown as average normalized % recovery ± SEM (see Supplementary Fig. 2a for representative FRAP image sequences). G3BP1 S149A -GFP shows much lower recovery than G3BP1 S149E -GFP; G3BP1-GFP is intermediate between G3BP1 S149A -GFP and G3BP1 S149E -GFP ( N ≥ 13 axons over 3 repetitions; * p ≤ 0.05 between G3BP1 S149A -GFP vs. G3BP1 S149E -GFP by one-way ANOVA with Tukey HSD post-hoc). Only the 0–320 s. recovery signals for GFP are shown (at 840 s. GFP showed 85.5 ± 4.7% recovery with p ≤ 0.0001 vs. G3BP1 S149E -GFP by one-way ANOVA with Tukey HSD post-hoc). d – e Exposure-matched confocal images for G3BP1 PS149 and NF are shown for sciatic nerve ( d ) as in Fig. 1e . There is a striking increase in G3BP1 PS149 immunoreactivity in the regenerating axons. Quantifications of these signals are shown as mean ± SEM ( e ; N = 3; * p ≤ 0.05 by one-way ANOVA with Tukey HSD post-hoc) [scale bar = 20 µm]. f – g Distal axons of cultured DRGs immunostained with pan-G3BP1 vs. G3BP1 PS149 antibodies are shown as indicated ( f ). Aggregates of G3BP1 are visible in the axon shaft (arrow), but decrease moving distally towards the growth cone (arrowhead). G3BP1 PS149 signals are fairly consistent and extend into the growth cone (arrowhead). Quantification of signals ( g ) shows significant increase in ratio of G3BP1 PS149 immunoreactivity to G3BP1 aggregates moving distally to the growth cone ( N ≥ 9 neurons each over 3 repetitions; * p ≤ 0.05 vs. 70–80 µm bin by one-way ANOVA with Tukey HSD post-hoc) [scale bar = 20 µm]

    Journal: Nature Communications

    Article Title: Axonal G3BP1 stress granule protein limits axonal mRNA translation and nerve regeneration

    doi: 10.1038/s41467-018-05647-x

    Figure Lengend Snippet: G3BP1 is phosphorylated in regenerating axons. a Representative images for axons of DRG neurons transfected with indicated G3BP1 constructs vs. eGFP are shown. G3BP1-GFP and G3BP1 S149A -GFP show prominent aggregates in axons that colocalize with HuR (arrows). In contrast, axonal signals for G3BP1 S149E -GFP and eGFP appear diffuse [scale bar = 5 µm]. b Quantification of axonal aggregates for G3BP1-GFP, G3BP1 S149A -GFP, and G3BP1 S149E -GFP is shown as average ± SEM ( N ≥ 10 neurons over 3 repetitions; *** p ≤ 0.005 by one-way ANOVA with Tukey HSD post-hoc). c FRAP analyses for neurons transfected with constructs as in A are shown as average normalized % recovery ± SEM (see Supplementary Fig. 2a for representative FRAP image sequences). G3BP1 S149A -GFP shows much lower recovery than G3BP1 S149E -GFP; G3BP1-GFP is intermediate between G3BP1 S149A -GFP and G3BP1 S149E -GFP ( N ≥ 13 axons over 3 repetitions; * p ≤ 0.05 between G3BP1 S149A -GFP vs. G3BP1 S149E -GFP by one-way ANOVA with Tukey HSD post-hoc). Only the 0–320 s. recovery signals for GFP are shown (at 840 s. GFP showed 85.5 ± 4.7% recovery with p ≤ 0.0001 vs. G3BP1 S149E -GFP by one-way ANOVA with Tukey HSD post-hoc). d – e Exposure-matched confocal images for G3BP1 PS149 and NF are shown for sciatic nerve ( d ) as in Fig. 1e . There is a striking increase in G3BP1 PS149 immunoreactivity in the regenerating axons. Quantifications of these signals are shown as mean ± SEM ( e ; N = 3; * p ≤ 0.05 by one-way ANOVA with Tukey HSD post-hoc) [scale bar = 20 µm]. f – g Distal axons of cultured DRGs immunostained with pan-G3BP1 vs. G3BP1 PS149 antibodies are shown as indicated ( f ). Aggregates of G3BP1 are visible in the axon shaft (arrow), but decrease moving distally towards the growth cone (arrowhead). G3BP1 PS149 signals are fairly consistent and extend into the growth cone (arrowhead). Quantification of signals ( g ) shows significant increase in ratio of G3BP1 PS149 immunoreactivity to G3BP1 aggregates moving distally to the growth cone ( N ≥ 9 neurons each over 3 repetitions; * p ≤ 0.05 vs. 70–80 µm bin by one-way ANOVA with Tukey HSD post-hoc) [scale bar = 20 µm]

    Article Snippet: Primary antibodies consisted of: rabbit anti-G3BP1 (1:200, Sigma), RT97 mouse anti-neurofilament (NF; 1:500, Devel.

    Techniques: Transfection, Construct, Cell Culture

    G3BP1 acidic domain expression accelerates nerve regeneration. a Schematic of G3BP1 domains as defined by Tourriere et al. (2003) 7 . b Representative images for NF-labeled DRG neurons transfected with indicated constructs are shown. Images were acquired at 60 h post-transfection [scale bar = 100 μm]. Axonal localization of these G3BP1-GFP domain proteins and quantitation of axon growth from G3BP1 domain-expressing DRG neurons are shown as Supplementary Fig. 4a-b . c Extent of axon regeneration at 7 d post sciatic nerve crush in adult rats transduced with AAV5 encoding G3BP1-BFP, G3BP1 B domain-BFP, G3BP1 D domain-BFP, or GFP control is shown as mean axonal profiles relative to crush site (0 mm) ± SEM. For representative images see Supplementary Fig. 5a ( N ≥ 5 animals per condition; * = p ≤ 0.05 and **** = p ≤ 0.0001 between B domain-BFP vs. GFP transduced animals by one-way ANOVA with Tukey HSD post-hoc). d Animals transduced with AAV5 encoding G3BP1 B domain-BFP vs. GFP were subjected to sciatic nerve crush and regeneration was assessed by muscle M response in tibialis anterior and gastrocnemius. Values are shown as average % intact M responses ± SEM (lines) with data points for individual animals plotted (* p ≤ 0.05 and ** p ≤ 0.01 for B domain vs. GFP by Student’s t -test for indicated data pairs). See Supplementary Fig. 5c for representative electrophysiological data. e Quantitation of axon growth from DRGs (left) and cortical neurons (right) treated with cell-permeable 168–189 or 190–208 G3BP1 peptides is shown. For DRGs, peptides were added to dissociated naive or 7 d injury-conditioned DRGs at 12 h and axon growth was assessed at 36 h in vitro. For cortical neurons, peptides were added to the axonal compartment of microfluidic devices at 3 d in vitro (DIV), and axon growth was assessed at 6 DIV. See Supplementary Fig. 6c for images of cortical cultures ( N ≥ 95 over 3 DRG cultures and 9 microfluidic devices over 3 cultures; *** p ≤ 0.005 by one-way ANOVA with Tukey HSD post-hoc)

    Journal: Nature Communications

    Article Title: Axonal G3BP1 stress granule protein limits axonal mRNA translation and nerve regeneration

    doi: 10.1038/s41467-018-05647-x

    Figure Lengend Snippet: G3BP1 acidic domain expression accelerates nerve regeneration. a Schematic of G3BP1 domains as defined by Tourriere et al. (2003) 7 . b Representative images for NF-labeled DRG neurons transfected with indicated constructs are shown. Images were acquired at 60 h post-transfection [scale bar = 100 μm]. Axonal localization of these G3BP1-GFP domain proteins and quantitation of axon growth from G3BP1 domain-expressing DRG neurons are shown as Supplementary Fig. 4a-b . c Extent of axon regeneration at 7 d post sciatic nerve crush in adult rats transduced with AAV5 encoding G3BP1-BFP, G3BP1 B domain-BFP, G3BP1 D domain-BFP, or GFP control is shown as mean axonal profiles relative to crush site (0 mm) ± SEM. For representative images see Supplementary Fig. 5a ( N ≥ 5 animals per condition; * = p ≤ 0.05 and **** = p ≤ 0.0001 between B domain-BFP vs. GFP transduced animals by one-way ANOVA with Tukey HSD post-hoc). d Animals transduced with AAV5 encoding G3BP1 B domain-BFP vs. GFP were subjected to sciatic nerve crush and regeneration was assessed by muscle M response in tibialis anterior and gastrocnemius. Values are shown as average % intact M responses ± SEM (lines) with data points for individual animals plotted (* p ≤ 0.05 and ** p ≤ 0.01 for B domain vs. GFP by Student’s t -test for indicated data pairs). See Supplementary Fig. 5c for representative electrophysiological data. e Quantitation of axon growth from DRGs (left) and cortical neurons (right) treated with cell-permeable 168–189 or 190–208 G3BP1 peptides is shown. For DRGs, peptides were added to dissociated naive or 7 d injury-conditioned DRGs at 12 h and axon growth was assessed at 36 h in vitro. For cortical neurons, peptides were added to the axonal compartment of microfluidic devices at 3 d in vitro (DIV), and axon growth was assessed at 6 DIV. See Supplementary Fig. 6c for images of cortical cultures ( N ≥ 95 over 3 DRG cultures and 9 microfluidic devices over 3 cultures; *** p ≤ 0.005 by one-way ANOVA with Tukey HSD post-hoc)

    Article Snippet: Primary antibodies consisted of: rabbit anti-G3BP1 (1:200, Sigma), RT97 mouse anti-neurofilament (NF; 1:500, Devel.

    Techniques: Expressing, Labeling, Transfection, Construct, Quantitation Assay, Transduction, In Vitro

    G3BP1 regulates translation of axonal mRNAs. a Images of FISH/IF for Nrn1 mRNA and G3BP1 protein are shown for axons of naive and 7 d injury-conditioned DRG neurons. Colocalization panel (Coloc) represents the mRNA:G3BP1 colocalization in a single optical plane (see Supplementary Fig. 3 for Impβ1 and Gap43 mRNA + G3BP1 colocalization images) [scale bar = 5 µm]. b Quantification of colocalizations for Nrn1, Impβ1 , and Gap43 mRNAs with G3BP1 in axons of neurons cultured from naive or 7 d injury-conditioned animals shown as average Pearson’s coefficient ± SEM ( N ≥ 21 neurons over 3 repetitions; ** p ≤ 0.01 and *** p ≤ 0.005 by one-way ANOVA with Tukey HSD post-hoc). c Schematics of translation reporter constructs used in panels d – h and Supplementary Movies 1 – 3 . d Representative FRAP image sequences for DRG neurons co-transfected with GFP MYR 5′/3′nrn1 plus BFP or G3BP1-BFP. Boxed regions represent the photobleached ROIs. Videos for these images are included as Supplementary Movie 1 [scale bar = 20 µm]. e – g Quantifications of FRAP assays from DRGs expressing GFP MYR 5′/3′nrn1 ( e ) or GFP MYR 5′/3′impβ1 ( f ) or mCh MYR 5′/3′gap43 translation reporters along with G3BP1-BFP or control BFP are shown as normalized, average % recovery ± SEM ( N ≥ 11 neurons over 3 repetitions; * p ≤ 0.05, and ** p ≤ 0.01 for BFP vs. G3BP1-BFP, # p ≤ 0.05, ## p ≤ 0.01, and #### p ≤ 0.0001 for BFP vs. translation inhibitors by one-way ANOVA with Tukey HSD post-hoc). Representative videos for these FRAP sequences are included as Supplementary Movies 1 – 3 . h HEK293T cells transfected with GFP MYR 5′/3′nrn1, GFP MYR 5′/3′ impβ1, and mCh MYR 5′/3′gap43 show significant enrichment of GFP MYR 5 ′ /3 ′ nrn1 and GFP MYR 5 ′ /3 ′ impβ1 mRNAs coimmunoprecipitating with G3BP1 vs. control ( N = 4 culture preparations; * p ≤ 0.05 by Student’s t -test). Western blot validating G3BP1 immunoprecipitation shown as inset. Values shown as average percent bound mRNA relative to input ± SEM

    Journal: Nature Communications

    Article Title: Axonal G3BP1 stress granule protein limits axonal mRNA translation and nerve regeneration

    doi: 10.1038/s41467-018-05647-x

    Figure Lengend Snippet: G3BP1 regulates translation of axonal mRNAs. a Images of FISH/IF for Nrn1 mRNA and G3BP1 protein are shown for axons of naive and 7 d injury-conditioned DRG neurons. Colocalization panel (Coloc) represents the mRNA:G3BP1 colocalization in a single optical plane (see Supplementary Fig. 3 for Impβ1 and Gap43 mRNA + G3BP1 colocalization images) [scale bar = 5 µm]. b Quantification of colocalizations for Nrn1, Impβ1 , and Gap43 mRNAs with G3BP1 in axons of neurons cultured from naive or 7 d injury-conditioned animals shown as average Pearson’s coefficient ± SEM ( N ≥ 21 neurons over 3 repetitions; ** p ≤ 0.01 and *** p ≤ 0.005 by one-way ANOVA with Tukey HSD post-hoc). c Schematics of translation reporter constructs used in panels d – h and Supplementary Movies 1 – 3 . d Representative FRAP image sequences for DRG neurons co-transfected with GFP MYR 5′/3′nrn1 plus BFP or G3BP1-BFP. Boxed regions represent the photobleached ROIs. Videos for these images are included as Supplementary Movie 1 [scale bar = 20 µm]. e – g Quantifications of FRAP assays from DRGs expressing GFP MYR 5′/3′nrn1 ( e ) or GFP MYR 5′/3′impβ1 ( f ) or mCh MYR 5′/3′gap43 translation reporters along with G3BP1-BFP or control BFP are shown as normalized, average % recovery ± SEM ( N ≥ 11 neurons over 3 repetitions; * p ≤ 0.05, and ** p ≤ 0.01 for BFP vs. G3BP1-BFP, # p ≤ 0.05, ## p ≤ 0.01, and #### p ≤ 0.0001 for BFP vs. translation inhibitors by one-way ANOVA with Tukey HSD post-hoc). Representative videos for these FRAP sequences are included as Supplementary Movies 1 – 3 . h HEK293T cells transfected with GFP MYR 5′/3′nrn1, GFP MYR 5′/3′ impβ1, and mCh MYR 5′/3′gap43 show significant enrichment of GFP MYR 5 ′ /3 ′ nrn1 and GFP MYR 5 ′ /3 ′ impβ1 mRNAs coimmunoprecipitating with G3BP1 vs. control ( N = 4 culture preparations; * p ≤ 0.05 by Student’s t -test). Western blot validating G3BP1 immunoprecipitation shown as inset. Values shown as average percent bound mRNA relative to input ± SEM

    Article Snippet: Primary antibodies consisted of: rabbit anti-G3BP1 (1:200, Sigma), RT97 mouse anti-neurofilament (NF; 1:500, Devel.

    Techniques: Fluorescence In Situ Hybridization, Cell Culture, Construct, Transfection, Expressing, Western Blot, Immunoprecipitation

    CCDC170 protein levels affect the rate of 2D cell migration. a. Live cell imaging was carried out to monitor migratory activity of MCF-7 cells expressing either GFP-CCDC170 or non-fused GFP as a negative control. Cell migration tracks (blue) were quantified with ImageJ software by tracing the center of the cells. b. Average migration distance between GFP vs. GFP-CCDC170 overexpressing cells were compared ( t -test, * P

    Journal: EBioMedicine

    Article Title: The Protein Encoded by the CCDC170 Breast Cancer Gene Functions to Organize the Golgi-Microtubule Network

    doi: 10.1016/j.ebiom.2017.06.024

    Figure Lengend Snippet: CCDC170 protein levels affect the rate of 2D cell migration. a. Live cell imaging was carried out to monitor migratory activity of MCF-7 cells expressing either GFP-CCDC170 or non-fused GFP as a negative control. Cell migration tracks (blue) were quantified with ImageJ software by tracing the center of the cells. b. Average migration distance between GFP vs. GFP-CCDC170 overexpressing cells were compared ( t -test, * P

    Article Snippet: The following primary antibodies were used for IF: AKAP9 rabbit antibody (1:100, #HPA008548, Sigma-Aldrich), CCDC170 rabbit antibody (1:100, HPA027114, Sigma-Aldrich).

    Techniques: Migration, Live Cell Imaging, Activity Assay, Expressing, Negative Control, Software

    MAP4, a candidate functional interaction partner of CCDC170 identified by BioID. a. MAP4 was detected as a candidate CCDC170 functional binding partner using BioID (Fig. S13, Tables S2 and S3). MAP4 is a microtubule-associated protein and plays a major role in the regulation of MT stabilization. To determine if MAP4 plays a role in CCDC170-mediated α-tubulin acetylation, HeLa cells were transfected with GFP-CCDC170 and after 20 h were treated with nocodazole for 4 h to promote MT disassembly. Cells were then immediately fixed and stained: GFP signal (green), MAP4 (red), and ac-α-tubulin (purple, upper panel) or total α-tubulin (purple, lower panel). Representative images show that in cells overexpressing GFP-CCDC170, perinuclear MTs become resistant to nocodazole-driven disassembly, and MAP4 becomes enriched and co-localized with CCDC170 at these perinuclear regions. b. Quantitative image analysis of MAP4 in GFP-CCDC170-expressing areas vs CCDC170-negative areas at perinuclear regions. Such quantitation was able to detect a significant difference in the MAP4 signal between CCDC170-positive and -negative areas at perinuclear regions ( n = 50 cells, * P

    Journal: EBioMedicine

    Article Title: The Protein Encoded by the CCDC170 Breast Cancer Gene Functions to Organize the Golgi-Microtubule Network

    doi: 10.1016/j.ebiom.2017.06.024

    Figure Lengend Snippet: MAP4, a candidate functional interaction partner of CCDC170 identified by BioID. a. MAP4 was detected as a candidate CCDC170 functional binding partner using BioID (Fig. S13, Tables S2 and S3). MAP4 is a microtubule-associated protein and plays a major role in the regulation of MT stabilization. To determine if MAP4 plays a role in CCDC170-mediated α-tubulin acetylation, HeLa cells were transfected with GFP-CCDC170 and after 20 h were treated with nocodazole for 4 h to promote MT disassembly. Cells were then immediately fixed and stained: GFP signal (green), MAP4 (red), and ac-α-tubulin (purple, upper panel) or total α-tubulin (purple, lower panel). Representative images show that in cells overexpressing GFP-CCDC170, perinuclear MTs become resistant to nocodazole-driven disassembly, and MAP4 becomes enriched and co-localized with CCDC170 at these perinuclear regions. b. Quantitative image analysis of MAP4 in GFP-CCDC170-expressing areas vs CCDC170-negative areas at perinuclear regions. Such quantitation was able to detect a significant difference in the MAP4 signal between CCDC170-positive and -negative areas at perinuclear regions ( n = 50 cells, * P

    Article Snippet: The following primary antibodies were used for IF: AKAP9 rabbit antibody (1:100, #HPA008548, Sigma-Aldrich), CCDC170 rabbit antibody (1:100, HPA027114, Sigma-Aldrich).

    Techniques: Functional Assay, Binding Assay, Transfection, Staining, Expressing, Quantitation Assay

    CCDC170 enhances MT stabilization. a and b. HeLa cells were transfected with GFP-CCDC170, and after 20 h were treated with nocodazole for 4 h to depolymerize MTs and observe potential CCDC170-induced resistance to nocodazole-induced MT depolymerization. Cells were then fixed and stained with anti-GFP and anti-α-tubulin (purple) antibodies 0 min after Nocodazole treatment (Noco WO 0 min) or 30 min after nocodazole washout to observe MT re-polymerization (Noco WO 30 min). As shown in panel d, after 4 h of nocodazole treatment, cells expressing GFP-CCDC170 (GFP-positive) showed nocodazole-resistant perinuclear MTs, as compared to neighboring nontransfected cells (GFP-negative). The GFP-CCDC170-induced nocodazole-resistance of MTs was quantitated by Corrected Total Cell Fluorescence (CTCF) counts (Panel b). Data represents absolute CTCF signal in GFP-positive versus GFP-negative cells ( n = 100; * P

    Journal: EBioMedicine

    Article Title: The Protein Encoded by the CCDC170 Breast Cancer Gene Functions to Organize the Golgi-Microtubule Network

    doi: 10.1016/j.ebiom.2017.06.024

    Figure Lengend Snippet: CCDC170 enhances MT stabilization. a and b. HeLa cells were transfected with GFP-CCDC170, and after 20 h were treated with nocodazole for 4 h to depolymerize MTs and observe potential CCDC170-induced resistance to nocodazole-induced MT depolymerization. Cells were then fixed and stained with anti-GFP and anti-α-tubulin (purple) antibodies 0 min after Nocodazole treatment (Noco WO 0 min) or 30 min after nocodazole washout to observe MT re-polymerization (Noco WO 30 min). As shown in panel d, after 4 h of nocodazole treatment, cells expressing GFP-CCDC170 (GFP-positive) showed nocodazole-resistant perinuclear MTs, as compared to neighboring nontransfected cells (GFP-negative). The GFP-CCDC170-induced nocodazole-resistance of MTs was quantitated by Corrected Total Cell Fluorescence (CTCF) counts (Panel b). Data represents absolute CTCF signal in GFP-positive versus GFP-negative cells ( n = 100; * P

    Article Snippet: The following primary antibodies were used for IF: AKAP9 rabbit antibody (1:100, #HPA008548, Sigma-Aldrich), CCDC170 rabbit antibody (1:100, HPA027114, Sigma-Aldrich).

    Techniques: Transfection, Staining, Expressing, Fluorescence

    The CCDC170/C6orf97 locus is associated with significant Differential Allele Specific Expression (DASE) and Genome Wide Association (GWA) signals. a. SNPs at the CCDC170/C6orf97-ESR1 locus that are associated with breast cancer and DASE. Upper plot shows regional GWA and DASE plots of the chromosome 6q25.1 loci CCDC170/C6orf97 and ESR1 . Results of Log 10 ( P -value) are shown for SNPs associated with breast cancer (red dots) for the region of 151.8–152.2 Mb. P -values plotted for SNPs reported by multiple studies are shown as average P -values. Results of DASE (green squares) at Log 2 (fold-mRNA expression differences from one allele to the other) are calculated and averaged for primary HMEC lines ( n = 30) by SNP-based analysis ( Gao et al., 2012 ). Gene locations and SNPs used for DASE analyses (lower map) are from the UCSC Genome Browser assembly. b. Results of DASE analysis of CCDC170 (in blue) or ESR1 (in red) at Log2 (fold-changes) are calculated by gene-based analysis in individual primary HMEC lines ( n = 30). DASE could not be calculated for all samples because the heterozygous DASE-detection SNPs were not available for some samples. c. DASE comparison between CCDC170 and ESR1 genes (upper panel: t -test; lower panel: Matched Pairs test).

    Journal: EBioMedicine

    Article Title: The Protein Encoded by the CCDC170 Breast Cancer Gene Functions to Organize the Golgi-Microtubule Network

    doi: 10.1016/j.ebiom.2017.06.024

    Figure Lengend Snippet: The CCDC170/C6orf97 locus is associated with significant Differential Allele Specific Expression (DASE) and Genome Wide Association (GWA) signals. a. SNPs at the CCDC170/C6orf97-ESR1 locus that are associated with breast cancer and DASE. Upper plot shows regional GWA and DASE plots of the chromosome 6q25.1 loci CCDC170/C6orf97 and ESR1 . Results of Log 10 ( P -value) are shown for SNPs associated with breast cancer (red dots) for the region of 151.8–152.2 Mb. P -values plotted for SNPs reported by multiple studies are shown as average P -values. Results of DASE (green squares) at Log 2 (fold-mRNA expression differences from one allele to the other) are calculated and averaged for primary HMEC lines ( n = 30) by SNP-based analysis ( Gao et al., 2012 ). Gene locations and SNPs used for DASE analyses (lower map) are from the UCSC Genome Browser assembly. b. Results of DASE analysis of CCDC170 (in blue) or ESR1 (in red) at Log2 (fold-changes) are calculated by gene-based analysis in individual primary HMEC lines ( n = 30). DASE could not be calculated for all samples because the heterozygous DASE-detection SNPs were not available for some samples. c. DASE comparison between CCDC170 and ESR1 genes (upper panel: t -test; lower panel: Matched Pairs test).

    Article Snippet: The following primary antibodies were used for IF: AKAP9 rabbit antibody (1:100, #HPA008548, Sigma-Aldrich), CCDC170 rabbit antibody (1:100, HPA027114, Sigma-Aldrich).

    Techniques: Expressing, GWAS

    CCDC170 overexpression promotes spreading or fragmentation of the Golgi ribbon structure. a. To examine whether overexpression of GFP-CCDC170 promoted reorganization of the Golgi, HeLa cells were transfected with non-fused GFP as a negative control, GFP-CCDC170, or clinical relevant truncation constructs (355–715 and 593–715). Quantitative results showed that CCDC170 overexpression can trigger spreading of the cis-Golgi ribbon structure as measured by both the fraction of CCDC170 positive cells showing perinuclear Golgi spreading (RCAS1 marker), as well as the angle of spreading of the cis-Golgi in individual cells (Ɵ). The diffusely expressed 593–715 fragment had no effect on Golgi spreading, while the 355–715 fragment showed an effect similar to the full length protein. b. 3D-rendering showing that CCDC170 overexpression triggers a dramatic spreading or fragmentation of the Golgi ribbon structure. c. Distribution of cells with different ranges of Golgi ribbon angle (Ɵ: 0°–360°), in cells expressing non-fused GFP, GFP-CCDC170, GFP-593-715 and GFP-355-715 [cell number ( n ) = 100 for each construct; vs. non-fused GFP (180°–360°); * P

    Journal: EBioMedicine

    Article Title: The Protein Encoded by the CCDC170 Breast Cancer Gene Functions to Organize the Golgi-Microtubule Network

    doi: 10.1016/j.ebiom.2017.06.024

    Figure Lengend Snippet: CCDC170 overexpression promotes spreading or fragmentation of the Golgi ribbon structure. a. To examine whether overexpression of GFP-CCDC170 promoted reorganization of the Golgi, HeLa cells were transfected with non-fused GFP as a negative control, GFP-CCDC170, or clinical relevant truncation constructs (355–715 and 593–715). Quantitative results showed that CCDC170 overexpression can trigger spreading of the cis-Golgi ribbon structure as measured by both the fraction of CCDC170 positive cells showing perinuclear Golgi spreading (RCAS1 marker), as well as the angle of spreading of the cis-Golgi in individual cells (Ɵ). The diffusely expressed 593–715 fragment had no effect on Golgi spreading, while the 355–715 fragment showed an effect similar to the full length protein. b. 3D-rendering showing that CCDC170 overexpression triggers a dramatic spreading or fragmentation of the Golgi ribbon structure. c. Distribution of cells with different ranges of Golgi ribbon angle (Ɵ: 0°–360°), in cells expressing non-fused GFP, GFP-CCDC170, GFP-593-715 and GFP-355-715 [cell number ( n ) = 100 for each construct; vs. non-fused GFP (180°–360°); * P

    Article Snippet: The following primary antibodies were used for IF: AKAP9 rabbit antibody (1:100, #HPA008548, Sigma-Aldrich), CCDC170 rabbit antibody (1:100, HPA027114, Sigma-Aldrich).

    Techniques: Over Expression, Transfection, Negative Control, Construct, Marker, Expressing

    CCDC170 localizes to the Golgi apparatus and has microtubule (MT)-binding activities. a. Subcellular localization of the GFP-CCDC170 fusion protein in HeLa cells. b. Top panel: Z -stack images were obtained using Leica SP8 confocal microscope. A 3D-rendering was created using IMARIS 7.5.0 software. Nucleus is shown in blue, with GFP-CCDC170 shown in green. The GFP-tagged protein localized to a membranous structure suspected to be the Golgi apparatus. Lower panel: Diagram of Golgi apparatus. c. GFP-CCDC170 Golgi localization was confirmed in HeLa cells by co-staining with an antibody to each of three resident Golgi proteins (in red): TGN46 (trans-Golgi), ManII (medial-Golgi), and RCAS1 (cis-Golgi). The nuclei are shown in blue (DAPI) as a reference. d. Subcellular localization of truncation mutants of N-terminally GFP-tagged CCDC170 in HeLa cells. All of these breast cancer-associated truncations of CCDC170 resulted in the loss of Golgi localization. Clinically relevant truncations are referenced from Cancer Genome Project and a recent report ( Veeraraghavan et al., 2014 ). e. CCDC170 associates with perinuclear MTs in HeLa cells. GFP-CCDC170 (green) and α-tubulin (purple) are shown. f. Upper panels: The WT GFP-CCDC170 was coexpressed with WT RFP-CCDC170 in HeLa cells. Bottom Panels: The GFP-CCDC170 (355–715) fragment was coexpressed with WT RFP-CCDC170 in HeLa cells. A dramatic relocalization of the wild type protein from the Golgi to MTs was observed. g. Upper panels: GFP CCDC170 1–649 fragment (green) expressed in HeLa cells localizes to MTs (red). Bottom panels: A comparison of transfected and untransfected cells in the same culture indicates that overexpression of the GFP CCDC170 1–649 fragment (green) can induce MT bundling (red). h. Diagram summarizing mapping of Golgi- and MT-association domains, based on truncated constructs. Additional data supporting these mapping studies are shown in Fig. S6.

    Journal: EBioMedicine

    Article Title: The Protein Encoded by the CCDC170 Breast Cancer Gene Functions to Organize the Golgi-Microtubule Network

    doi: 10.1016/j.ebiom.2017.06.024

    Figure Lengend Snippet: CCDC170 localizes to the Golgi apparatus and has microtubule (MT)-binding activities. a. Subcellular localization of the GFP-CCDC170 fusion protein in HeLa cells. b. Top panel: Z -stack images were obtained using Leica SP8 confocal microscope. A 3D-rendering was created using IMARIS 7.5.0 software. Nucleus is shown in blue, with GFP-CCDC170 shown in green. The GFP-tagged protein localized to a membranous structure suspected to be the Golgi apparatus. Lower panel: Diagram of Golgi apparatus. c. GFP-CCDC170 Golgi localization was confirmed in HeLa cells by co-staining with an antibody to each of three resident Golgi proteins (in red): TGN46 (trans-Golgi), ManII (medial-Golgi), and RCAS1 (cis-Golgi). The nuclei are shown in blue (DAPI) as a reference. d. Subcellular localization of truncation mutants of N-terminally GFP-tagged CCDC170 in HeLa cells. All of these breast cancer-associated truncations of CCDC170 resulted in the loss of Golgi localization. Clinically relevant truncations are referenced from Cancer Genome Project and a recent report ( Veeraraghavan et al., 2014 ). e. CCDC170 associates with perinuclear MTs in HeLa cells. GFP-CCDC170 (green) and α-tubulin (purple) are shown. f. Upper panels: The WT GFP-CCDC170 was coexpressed with WT RFP-CCDC170 in HeLa cells. Bottom Panels: The GFP-CCDC170 (355–715) fragment was coexpressed with WT RFP-CCDC170 in HeLa cells. A dramatic relocalization of the wild type protein from the Golgi to MTs was observed. g. Upper panels: GFP CCDC170 1–649 fragment (green) expressed in HeLa cells localizes to MTs (red). Bottom panels: A comparison of transfected and untransfected cells in the same culture indicates that overexpression of the GFP CCDC170 1–649 fragment (green) can induce MT bundling (red). h. Diagram summarizing mapping of Golgi- and MT-association domains, based on truncated constructs. Additional data supporting these mapping studies are shown in Fig. S6.

    Article Snippet: The following primary antibodies were used for IF: AKAP9 rabbit antibody (1:100, #HPA008548, Sigma-Aldrich), CCDC170 rabbit antibody (1:100, HPA027114, Sigma-Aldrich).

    Techniques: Binding Assay, Microscopy, Software, Staining, Transfection, Over Expression, Construct

    CCDC170 enhances alpha-tubulin acetylation. a. HeLa cells were transiently transfected with GFP-CCDC170 full length, the clinically relevant N-terminally truncated forms (GFP-593-715, GFP-355-715) or a non-fused GFP negative control, and were subjected to immunofluorescence for acetylated alpha-tubulin (ac-α-tubulin) (red). b. Corrected Total Cell Fluorescence (CTCF) counts from panel a. Data represent CTCF ratio of red signal (ac-α-tubulin) between GFP-positive and GFP-negative cells (ratio of red signal in GFP Positive: GFP Negative) as compared to the negative control of non-fused GFP ( n = 100, * P

    Journal: EBioMedicine

    Article Title: The Protein Encoded by the CCDC170 Breast Cancer Gene Functions to Organize the Golgi-Microtubule Network

    doi: 10.1016/j.ebiom.2017.06.024

    Figure Lengend Snippet: CCDC170 enhances alpha-tubulin acetylation. a. HeLa cells were transiently transfected with GFP-CCDC170 full length, the clinically relevant N-terminally truncated forms (GFP-593-715, GFP-355-715) or a non-fused GFP negative control, and were subjected to immunofluorescence for acetylated alpha-tubulin (ac-α-tubulin) (red). b. Corrected Total Cell Fluorescence (CTCF) counts from panel a. Data represent CTCF ratio of red signal (ac-α-tubulin) between GFP-positive and GFP-negative cells (ratio of red signal in GFP Positive: GFP Negative) as compared to the negative control of non-fused GFP ( n = 100, * P

    Article Snippet: The following primary antibodies were used for IF: AKAP9 rabbit antibody (1:100, #HPA008548, Sigma-Aldrich), CCDC170 rabbit antibody (1:100, HPA027114, Sigma-Aldrich).

    Techniques: Transfection, Negative Control, Immunofluorescence, Fluorescence

    CKAP2 plays a role in maintaining chromosome stability. (A–C) Mitotic cells were treated with colcemid in order to obtain metaphase spreads. Chromosome content was determined by counting the individual chromosomes in at least 100 metaphases. The results are presented as radial plots, where the concentric circle represents the relative ploidy and each symbol represents an individual cell. In parallel, here indicated are karyotypes analyzed by SKY showing the increased level of aneuploidy and chromosome instability in CKAP2-depleted cells.

    Journal: PLoS ONE

    Article Title: CKAP2 Ensures Chromosomal Stability by Maintaining the Integrity of Microtubule Nucleation Sites

    doi: 10.1371/journal.pone.0064575

    Figure Lengend Snippet: CKAP2 plays a role in maintaining chromosome stability. (A–C) Mitotic cells were treated with colcemid in order to obtain metaphase spreads. Chromosome content was determined by counting the individual chromosomes in at least 100 metaphases. The results are presented as radial plots, where the concentric circle represents the relative ploidy and each symbol represents an individual cell. In parallel, here indicated are karyotypes analyzed by SKY showing the increased level of aneuploidy and chromosome instability in CKAP2-depleted cells.

    Article Snippet: The antibodies used were mouse anti-CKAP2 (Abcam, 1∶1,000), rabbit anti-CKAP2 (Sigma-Aldrich, 1∶1,000), rat anti-α-tubulin (YL1/2) (Accu-Specs, 1∶1,000), mouse anti-phospho-histone H3 (Ser10, 6G3) (Cell Signaling, Danvers, MA, 1∶1,000), mouse anti-cyclin B1 (V152) (Cell Signaling, 1∶2,000), α/γ-tubulin (Cell Signaling, 1∶2,000), GAPDH (Sigma-Aldrich, 1∶40,000), and NuMA (BD Biosciences, 1∶2,000).

    Techniques:

    CKAP2-depleted cells show increased chromosome missegregation. (A) Control (shCTL) and CKAP2-depleted (shCKAP2) cells were transfected with histone H2B-Cherry constructs, selected with geneticin (G418), and analyzed with live-cell imaging. The movie shows CKAP2-depleted histone H2B-Cherry positive cells undergoing aberrant mitosis with chromosome missegregation resulting in two daughter nuclei with micronuclei. Arrows indicate lagging chromosomes and resultant micronuclei. (B) CKAP2-depleted cells were immunostained for Hec1 (green), α-tubulin (red), and merged with DAPI (blue). Cells with lagging chromosomes in anaphase and telophase were analyzed for merotelic attachments. Representative images of lagging chromosomes in anaphase and telophase are shown here. Magnified views emphasize merotelic attachments in lagging chromosomes. (C) The histogram represents the number of chromosome missegregation events for each histone H2B-Cherry positive experimental group. (D) Asynchronous shCTL and CKAP2-depleted cells were analyzed for evidence of chromosome missegregation, including micronuclei, nuclear blebs, and anaphase bridges. The results are plotted as the mean ± SD.

    Journal: PLoS ONE

    Article Title: CKAP2 Ensures Chromosomal Stability by Maintaining the Integrity of Microtubule Nucleation Sites

    doi: 10.1371/journal.pone.0064575

    Figure Lengend Snippet: CKAP2-depleted cells show increased chromosome missegregation. (A) Control (shCTL) and CKAP2-depleted (shCKAP2) cells were transfected with histone H2B-Cherry constructs, selected with geneticin (G418), and analyzed with live-cell imaging. The movie shows CKAP2-depleted histone H2B-Cherry positive cells undergoing aberrant mitosis with chromosome missegregation resulting in two daughter nuclei with micronuclei. Arrows indicate lagging chromosomes and resultant micronuclei. (B) CKAP2-depleted cells were immunostained for Hec1 (green), α-tubulin (red), and merged with DAPI (blue). Cells with lagging chromosomes in anaphase and telophase were analyzed for merotelic attachments. Representative images of lagging chromosomes in anaphase and telophase are shown here. Magnified views emphasize merotelic attachments in lagging chromosomes. (C) The histogram represents the number of chromosome missegregation events for each histone H2B-Cherry positive experimental group. (D) Asynchronous shCTL and CKAP2-depleted cells were analyzed for evidence of chromosome missegregation, including micronuclei, nuclear blebs, and anaphase bridges. The results are plotted as the mean ± SD.

    Article Snippet: The antibodies used were mouse anti-CKAP2 (Abcam, 1∶1,000), rabbit anti-CKAP2 (Sigma-Aldrich, 1∶1,000), rat anti-α-tubulin (YL1/2) (Accu-Specs, 1∶1,000), mouse anti-phospho-histone H3 (Ser10, 6G3) (Cell Signaling, Danvers, MA, 1∶1,000), mouse anti-cyclin B1 (V152) (Cell Signaling, 1∶2,000), α/γ-tubulin (Cell Signaling, 1∶2,000), GAPDH (Sigma-Aldrich, 1∶40,000), and NuMA (BD Biosciences, 1∶2,000).

    Techniques: Transfection, Construct, Live Cell Imaging

    CKAP2 is required for bipolar spindle assembly. (A) Representative images of control (shCTL) and CKAP2-depleted (shCKAP2) cells co-immunstained with CKAP2 (green), α-tubulin (red), and merged with DAPI (blue) (Scale bar: 2 µm). The data is presented as the mean green intensity per experiment group. One hundred images were analyzed per experimental group. (B) Mitotic cells in asysnchronous populations of control (shCTL) and CKAP2-depleted (shCKAP2) cells were analyzed for mitotic defects by co-immunostaining with γ-tubulin (green) and α-tubulin (red). Representative images of multipolar spindles were observed CKAP2-depleted cells. Over 100 spindles per experimental group were analyzed in two independent experiments. The results are presented as mean ± SD (Scale bar: 2 µm). P-values were determined using the Student’s t-test. (C) Thirty nuclei were analyzed to assess the percentage of cells with supernumerary centrosomes in CKAP2-depleted cells compared to controls.

    Journal: PLoS ONE

    Article Title: CKAP2 Ensures Chromosomal Stability by Maintaining the Integrity of Microtubule Nucleation Sites

    doi: 10.1371/journal.pone.0064575

    Figure Lengend Snippet: CKAP2 is required for bipolar spindle assembly. (A) Representative images of control (shCTL) and CKAP2-depleted (shCKAP2) cells co-immunstained with CKAP2 (green), α-tubulin (red), and merged with DAPI (blue) (Scale bar: 2 µm). The data is presented as the mean green intensity per experiment group. One hundred images were analyzed per experimental group. (B) Mitotic cells in asysnchronous populations of control (shCTL) and CKAP2-depleted (shCKAP2) cells were analyzed for mitotic defects by co-immunostaining with γ-tubulin (green) and α-tubulin (red). Representative images of multipolar spindles were observed CKAP2-depleted cells. Over 100 spindles per experimental group were analyzed in two independent experiments. The results are presented as mean ± SD (Scale bar: 2 µm). P-values were determined using the Student’s t-test. (C) Thirty nuclei were analyzed to assess the percentage of cells with supernumerary centrosomes in CKAP2-depleted cells compared to controls.

    Article Snippet: The antibodies used were mouse anti-CKAP2 (Abcam, 1∶1,000), rabbit anti-CKAP2 (Sigma-Aldrich, 1∶1,000), rat anti-α-tubulin (YL1/2) (Accu-Specs, 1∶1,000), mouse anti-phospho-histone H3 (Ser10, 6G3) (Cell Signaling, Danvers, MA, 1∶1,000), mouse anti-cyclin B1 (V152) (Cell Signaling, 1∶2,000), α/γ-tubulin (Cell Signaling, 1∶2,000), GAPDH (Sigma-Aldrich, 1∶40,000), and NuMA (BD Biosciences, 1∶2,000).

    Techniques: Immunostaining

    CKAP2 is required for anchoring of centrosome-nucleated microtubules to the spindle pole. (A) Nucleation was assessed after treating cells with 10 µg/ml nocodazole for 30 minutes and released into fresh media for 2, 30, and 60 minutes. shCTL and CKAP2-depleted cells were co-immunostained with γ-tubulin (green), Tyr-tubulin (red), and merged with DAPI (blue). (B) Two minutes post-nocodazole release, a cage-like structure was often observed in CKAP2-depleted cells. Representative images for each experimental group are shown. (C) Thirty minutes post-nocodazole release, microtubules are tethered at distinct poles, often with an increase cells with multipolar spindle poles in CKAP2-depleted cells. Representative images for each experimental group are shown. (D) Sixty minutes post nocodazole block, both the control and CKAP2-depleted cells have structured bipolar assembly. Representative images for each experimental group are shown. (E) Quantification of the shCTL cells with non-centrosomal α-tubulin staining at 2, 30, and 60 minutes is shown. Approximately 50 cells were counted per condition. (F) Measurements of the total γ-tubulin in both the centrosomes and the spindle pole area for CKAP2-depleted cells and controls. Y-axis indicates signal intensity units for γ-tubulin.

    Journal: PLoS ONE

    Article Title: CKAP2 Ensures Chromosomal Stability by Maintaining the Integrity of Microtubule Nucleation Sites

    doi: 10.1371/journal.pone.0064575

    Figure Lengend Snippet: CKAP2 is required for anchoring of centrosome-nucleated microtubules to the spindle pole. (A) Nucleation was assessed after treating cells with 10 µg/ml nocodazole for 30 minutes and released into fresh media for 2, 30, and 60 minutes. shCTL and CKAP2-depleted cells were co-immunostained with γ-tubulin (green), Tyr-tubulin (red), and merged with DAPI (blue). (B) Two minutes post-nocodazole release, a cage-like structure was often observed in CKAP2-depleted cells. Representative images for each experimental group are shown. (C) Thirty minutes post-nocodazole release, microtubules are tethered at distinct poles, often with an increase cells with multipolar spindle poles in CKAP2-depleted cells. Representative images for each experimental group are shown. (D) Sixty minutes post nocodazole block, both the control and CKAP2-depleted cells have structured bipolar assembly. Representative images for each experimental group are shown. (E) Quantification of the shCTL cells with non-centrosomal α-tubulin staining at 2, 30, and 60 minutes is shown. Approximately 50 cells were counted per condition. (F) Measurements of the total γ-tubulin in both the centrosomes and the spindle pole area for CKAP2-depleted cells and controls. Y-axis indicates signal intensity units for γ-tubulin.

    Article Snippet: The antibodies used were mouse anti-CKAP2 (Abcam, 1∶1,000), rabbit anti-CKAP2 (Sigma-Aldrich, 1∶1,000), rat anti-α-tubulin (YL1/2) (Accu-Specs, 1∶1,000), mouse anti-phospho-histone H3 (Ser10, 6G3) (Cell Signaling, Danvers, MA, 1∶1,000), mouse anti-cyclin B1 (V152) (Cell Signaling, 1∶2,000), α/γ-tubulin (Cell Signaling, 1∶2,000), GAPDH (Sigma-Aldrich, 1∶40,000), and NuMA (BD Biosciences, 1∶2,000).

    Techniques: Blocking Assay, Staining

    Centrosome nucleation capacity is unaffected in CKAP2-depleted cells. (A) Two minutes post-nocodazole release shCTL and CKAP2-depleted cells were co-immunostained with α-tubulin (DM1A) (green), pericentrin (red), and merged with DAPI (blue). One hundred cells with non-centrosomal tubulin staining were measured per experimental group. As already demonstrated, a cage-like structure was observed two minutes post-nocodazole release. Representative images for each experimental group are shown. (B) Nucleation capacity was determined by measuring the mean of α-tubulin fluorescence for both the control and CKAP2-depleted cells.

    Journal: PLoS ONE

    Article Title: CKAP2 Ensures Chromosomal Stability by Maintaining the Integrity of Microtubule Nucleation Sites

    doi: 10.1371/journal.pone.0064575

    Figure Lengend Snippet: Centrosome nucleation capacity is unaffected in CKAP2-depleted cells. (A) Two minutes post-nocodazole release shCTL and CKAP2-depleted cells were co-immunostained with α-tubulin (DM1A) (green), pericentrin (red), and merged with DAPI (blue). One hundred cells with non-centrosomal tubulin staining were measured per experimental group. As already demonstrated, a cage-like structure was observed two minutes post-nocodazole release. Representative images for each experimental group are shown. (B) Nucleation capacity was determined by measuring the mean of α-tubulin fluorescence for both the control and CKAP2-depleted cells.

    Article Snippet: The antibodies used were mouse anti-CKAP2 (Abcam, 1∶1,000), rabbit anti-CKAP2 (Sigma-Aldrich, 1∶1,000), rat anti-α-tubulin (YL1/2) (Accu-Specs, 1∶1,000), mouse anti-phospho-histone H3 (Ser10, 6G3) (Cell Signaling, Danvers, MA, 1∶1,000), mouse anti-cyclin B1 (V152) (Cell Signaling, 1∶2,000), α/γ-tubulin (Cell Signaling, 1∶2,000), GAPDH (Sigma-Aldrich, 1∶40,000), and NuMA (BD Biosciences, 1∶2,000).

    Techniques: Staining, Fluorescence

    Localization of CKAP2 at the spindle pole in human colorectal cancer cell line DLD-1. (A) DLD-1 cells co-immunostained with CKAP2 (green), α-tubulin (red), and DAPI (blue) depicting localization of CKAP2 to the spindle pole (Scale bar: 2 µm) (B) DLD-1 cells co-immunostained with CKAP2 (red), γ-tubulin (green) and DAPI (blue) demonstrating CKAP2 does not localize within the centrosome (Scale bar: 2 µm). (C) Mitotic cells were enriched by mitotic shake-off, lysed in hypotonic buffer, and the lysate fractionated by centrifugation. The pellet contains DNA, microtubules, and microtubule-associated proteins, whereas the supernatant contains the remaining proteins. These fractions were analyzed by immunoblot with antibodies specific to CKAP2, α/γ-tubulin, and γ-tubulin. The presence of CKAP2 in the pellet in both wild-type and nocodazole treated cells suggests that CKAP2 is indirectly associated with the mitotic spindle. (D) DLD-1 cells were transfected with shRNA, selected with puromycin, and single-cell separated by FACS based on GFP-positivity. Separated cells were synchronized overnight with nocodazole and harvested for immunoblot analysis with antibodies specific for CKAP2 and GAPDH. (E) To measure the affect of CKAP2 reduction on cell proliferation, populations of control (shCTL) and CKAP2-depleted cells (shCKAP2) were counted for six days and plotted. No significant differences in growth activity are observed.

    Journal: PLoS ONE

    Article Title: CKAP2 Ensures Chromosomal Stability by Maintaining the Integrity of Microtubule Nucleation Sites

    doi: 10.1371/journal.pone.0064575

    Figure Lengend Snippet: Localization of CKAP2 at the spindle pole in human colorectal cancer cell line DLD-1. (A) DLD-1 cells co-immunostained with CKAP2 (green), α-tubulin (red), and DAPI (blue) depicting localization of CKAP2 to the spindle pole (Scale bar: 2 µm) (B) DLD-1 cells co-immunostained with CKAP2 (red), γ-tubulin (green) and DAPI (blue) demonstrating CKAP2 does not localize within the centrosome (Scale bar: 2 µm). (C) Mitotic cells were enriched by mitotic shake-off, lysed in hypotonic buffer, and the lysate fractionated by centrifugation. The pellet contains DNA, microtubules, and microtubule-associated proteins, whereas the supernatant contains the remaining proteins. These fractions were analyzed by immunoblot with antibodies specific to CKAP2, α/γ-tubulin, and γ-tubulin. The presence of CKAP2 in the pellet in both wild-type and nocodazole treated cells suggests that CKAP2 is indirectly associated with the mitotic spindle. (D) DLD-1 cells were transfected with shRNA, selected with puromycin, and single-cell separated by FACS based on GFP-positivity. Separated cells were synchronized overnight with nocodazole and harvested for immunoblot analysis with antibodies specific for CKAP2 and GAPDH. (E) To measure the affect of CKAP2 reduction on cell proliferation, populations of control (shCTL) and CKAP2-depleted cells (shCKAP2) were counted for six days and plotted. No significant differences in growth activity are observed.

    Article Snippet: The antibodies used were mouse anti-CKAP2 (Abcam, 1∶1,000), rabbit anti-CKAP2 (Sigma-Aldrich, 1∶1,000), rat anti-α-tubulin (YL1/2) (Accu-Specs, 1∶1,000), mouse anti-phospho-histone H3 (Ser10, 6G3) (Cell Signaling, Danvers, MA, 1∶1,000), mouse anti-cyclin B1 (V152) (Cell Signaling, 1∶2,000), α/γ-tubulin (Cell Signaling, 1∶2,000), GAPDH (Sigma-Aldrich, 1∶40,000), and NuMA (BD Biosciences, 1∶2,000).

    Techniques: Centrifugation, Transfection, shRNA, FACS, Activity Assay

    NuMA expression and localization is not affected by CKAP2-depletion. (A) shCTL and shCKAP2 transfected cells were co-immunostained with NuMA (green) and α-tubulin (red), and merged with DAPI (blue). The expression of NuMA was confined to the spindle pole. Representative metaphase images show that the localization of NuMA remains intact. (B) Immunoblot analysis with antibodies specific for NuMA and GAPDH showed that the amount of NuMA protein was maintained despite the silencing of CKAP2. (C) shCTL and CKAP2-depleted cells were co-immunostained with NuMA (green), CKAP2 (red), and merged with DAPI (blue) showing only partial overlay between the two protein although they both are located at the spindle pole. (D) shCTL and CKAP2-depleted cells were synchronized with nocodazole, and after two minutes post release cells were co-immunostained with NuMA (green) and α-tubulin (red). Co-localization of NuMA and α-tubulin is shown in the cage-like structures in CKAP2-depleted cells, but not in control cells.

    Journal: PLoS ONE

    Article Title: CKAP2 Ensures Chromosomal Stability by Maintaining the Integrity of Microtubule Nucleation Sites

    doi: 10.1371/journal.pone.0064575

    Figure Lengend Snippet: NuMA expression and localization is not affected by CKAP2-depletion. (A) shCTL and shCKAP2 transfected cells were co-immunostained with NuMA (green) and α-tubulin (red), and merged with DAPI (blue). The expression of NuMA was confined to the spindle pole. Representative metaphase images show that the localization of NuMA remains intact. (B) Immunoblot analysis with antibodies specific for NuMA and GAPDH showed that the amount of NuMA protein was maintained despite the silencing of CKAP2. (C) shCTL and CKAP2-depleted cells were co-immunostained with NuMA (green), CKAP2 (red), and merged with DAPI (blue) showing only partial overlay between the two protein although they both are located at the spindle pole. (D) shCTL and CKAP2-depleted cells were synchronized with nocodazole, and after two minutes post release cells were co-immunostained with NuMA (green) and α-tubulin (red). Co-localization of NuMA and α-tubulin is shown in the cage-like structures in CKAP2-depleted cells, but not in control cells.

    Article Snippet: The antibodies used were mouse anti-CKAP2 (Abcam, 1∶1,000), rabbit anti-CKAP2 (Sigma-Aldrich, 1∶1,000), rat anti-α-tubulin (YL1/2) (Accu-Specs, 1∶1,000), mouse anti-phospho-histone H3 (Ser10, 6G3) (Cell Signaling, Danvers, MA, 1∶1,000), mouse anti-cyclin B1 (V152) (Cell Signaling, 1∶2,000), α/γ-tubulin (Cell Signaling, 1∶2,000), GAPDH (Sigma-Aldrich, 1∶40,000), and NuMA (BD Biosciences, 1∶2,000).

    Techniques: Expressing, Transfection

    Spindle pole defects in CKAP2-depleted cells. (A) Mitosis in an asynchronous population of control (shCTL) and CKAP2-depleted (shCKAP2) cells were co-immunostained with γ-tubulin (green), α-tubulin (red), and merged with DAPI (blue). The dispersal of γ-tubulin away from the centrosome and dislocation of the centrosome from the spindle pole was analyzed in 200 cells per experimental group in two independent experiments. Representative images for each experimental group and the mitotic defect are shown (Scale bar: 2 µm). Representative images for each experimental group are shown. (B) Quantification of the cells with dispersed γ-tubulin was presented as mean ± SD. P-values were determined using the Student’s t-test. (C) Quantification of the cells with the centrosome dislocated from the spindle pole was presented as mean ± SD. P-values were determined using the Student’s t-test (D) Spindle length measured in 50 mitotic cells with bipolar spindles in both controls and CKAP2-depleted cells is shown. P-value was determined by Student’s t-test. (E) Analysis of the number of misaligned chromosomes in bipolar metaphases shows statistical significant difference between control and CKAP2-depleted cells. More than 200 cells were counted per condition. P-value was determined by Student’s t-test.

    Journal: PLoS ONE

    Article Title: CKAP2 Ensures Chromosomal Stability by Maintaining the Integrity of Microtubule Nucleation Sites

    doi: 10.1371/journal.pone.0064575

    Figure Lengend Snippet: Spindle pole defects in CKAP2-depleted cells. (A) Mitosis in an asynchronous population of control (shCTL) and CKAP2-depleted (shCKAP2) cells were co-immunostained with γ-tubulin (green), α-tubulin (red), and merged with DAPI (blue). The dispersal of γ-tubulin away from the centrosome and dislocation of the centrosome from the spindle pole was analyzed in 200 cells per experimental group in two independent experiments. Representative images for each experimental group and the mitotic defect are shown (Scale bar: 2 µm). Representative images for each experimental group are shown. (B) Quantification of the cells with dispersed γ-tubulin was presented as mean ± SD. P-values were determined using the Student’s t-test. (C) Quantification of the cells with the centrosome dislocated from the spindle pole was presented as mean ± SD. P-values were determined using the Student’s t-test (D) Spindle length measured in 50 mitotic cells with bipolar spindles in both controls and CKAP2-depleted cells is shown. P-value was determined by Student’s t-test. (E) Analysis of the number of misaligned chromosomes in bipolar metaphases shows statistical significant difference between control and CKAP2-depleted cells. More than 200 cells were counted per condition. P-value was determined by Student’s t-test.

    Article Snippet: The antibodies used were mouse anti-CKAP2 (Abcam, 1∶1,000), rabbit anti-CKAP2 (Sigma-Aldrich, 1∶1,000), rat anti-α-tubulin (YL1/2) (Accu-Specs, 1∶1,000), mouse anti-phospho-histone H3 (Ser10, 6G3) (Cell Signaling, Danvers, MA, 1∶1,000), mouse anti-cyclin B1 (V152) (Cell Signaling, 1∶2,000), α/γ-tubulin (Cell Signaling, 1∶2,000), GAPDH (Sigma-Aldrich, 1∶40,000), and NuMA (BD Biosciences, 1∶2,000).

    Techniques:

    Live-cell imaging of WT PCSK9, PCSK9-ΔCTD-mC and LDLR-EGFP trafficking. HepG2 cells were co-transfected with LDLR-EGFP together with (A) WT PCSK9-mC (see S3 Video ) or (B) WT PCSK9-ΔCTD (see S4 Video ). Images were extracted from confocal microscopy time-lapse movies. Data are representative of at least three independent experiments. Scale bar , 20 μm.

    Journal: PLoS ONE

    Article Title: Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain

    doi: 10.1371/journal.pone.0157230

    Figure Lengend Snippet: Live-cell imaging of WT PCSK9, PCSK9-ΔCTD-mC and LDLR-EGFP trafficking. HepG2 cells were co-transfected with LDLR-EGFP together with (A) WT PCSK9-mC (see S3 Video ) or (B) WT PCSK9-ΔCTD (see S4 Video ). Images were extracted from confocal microscopy time-lapse movies. Data are representative of at least three independent experiments. Scale bar , 20 μm.

    Article Snippet: The cells were then incubated for 30 min with 1% BSA (Fraction V; Cat. #BP1605, Sigma) containing or not 0.1% Triton X-100, followed by overnight incubation at 4°C with rabbit anti-human PCSK9 (1:250), mouse anti-V5 (1/500; Cat. #R960-025, Life Technologies) and mouse anti-Golgin-97 (1/500; Cat. #sc-59820, Santa Cruz Biotechnology).

    Techniques: Live Cell Imaging, Transfection, Confocal Microscopy

    Lysosomal targeting of PCSK9-ΔCTD bypasses the need of CTD to induce LDLR degradation. (A-B) HepG2 cells were transfected without (Empty) or with V5-tagged PCSK9 full-length (FL), ΔCTD, CTD alone or PCSK9-TM-CT-Lamp1 chimeras (FL, F379A, ΔCTD, CTD) or Timp1-TM-CT-Lamp1 (herein used as control). Forty-eight hours post-transfection, LDLR, PCSK9 and β-actin (loading control) protein levels were analyzed by IB. Data are representative of at least three independent experiments

    Journal: PLoS ONE

    Article Title: Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain

    doi: 10.1371/journal.pone.0157230

    Figure Lengend Snippet: Lysosomal targeting of PCSK9-ΔCTD bypasses the need of CTD to induce LDLR degradation. (A-B) HepG2 cells were transfected without (Empty) or with V5-tagged PCSK9 full-length (FL), ΔCTD, CTD alone or PCSK9-TM-CT-Lamp1 chimeras (FL, F379A, ΔCTD, CTD) or Timp1-TM-CT-Lamp1 (herein used as control). Forty-eight hours post-transfection, LDLR, PCSK9 and β-actin (loading control) protein levels were analyzed by IB. Data are representative of at least three independent experiments

    Article Snippet: The cells were then incubated for 30 min with 1% BSA (Fraction V; Cat. #BP1605, Sigma) containing or not 0.1% Triton X-100, followed by overnight incubation at 4°C with rabbit anti-human PCSK9 (1:250), mouse anti-V5 (1/500; Cat. #R960-025, Life Technologies) and mouse anti-Golgin-97 (1/500; Cat. #sc-59820, Santa Cruz Biotechnology).

    Techniques: Transfection

    Live-cell imaging of PCSK9-D374Y-mC and LDLR-EGFP trafficking. HepG2 cells were transfected with LDLR-EGFP together with D374Y PCSK9-mC (A; transfected cells, see also S1 Video ) or incubated with PCSK9 D374Y-mC conditioned media (B; extracellular pathway, see also S2 Video ). Images were extracted from confocal microscopy time-lapse movies. Data are representative of at least three independent experiments. Scale bar , 20 μm.

    Journal: PLoS ONE

    Article Title: Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain

    doi: 10.1371/journal.pone.0157230

    Figure Lengend Snippet: Live-cell imaging of PCSK9-D374Y-mC and LDLR-EGFP trafficking. HepG2 cells were transfected with LDLR-EGFP together with D374Y PCSK9-mC (A; transfected cells, see also S1 Video ) or incubated with PCSK9 D374Y-mC conditioned media (B; extracellular pathway, see also S2 Video ). Images were extracted from confocal microscopy time-lapse movies. Data are representative of at least three independent experiments. Scale bar , 20 μm.

    Article Snippet: The cells were then incubated for 30 min with 1% BSA (Fraction V; Cat. #BP1605, Sigma) containing or not 0.1% Triton X-100, followed by overnight incubation at 4°C with rabbit anti-human PCSK9 (1:250), mouse anti-V5 (1/500; Cat. #R960-025, Life Technologies) and mouse anti-Golgin-97 (1/500; Cat. #sc-59820, Santa Cruz Biotechnology).

    Techniques: Live Cell Imaging, Transfection, Incubation, Confocal Microscopy

    Effect of PCSK9 natural mutants on TGN localization and trafficking dynamics by FRAP and iFRAP. (A) HepG2 cells were transfected with PCSK9-mC WT, GOF S127R, D129G, D374Y or LOF R46L mutants (red) and TGN localization was determined by colocalization (arrows) with Golgin-97 (green) and analysis by confocal microscopy. Nuclei were labeled with TO-PRO-3 Iodide (cyan). (B) Half time (T 1/2 ; min) and mobile fraction (%) calculated from FRAP (ER ➜ Golgi) and iFRAP (Golgi ➜ plasma membrane) experiments in living HepG2 cells for corresponding PCSK9-mC constructs shown in (A). Dashed square represent typical TGN localization of PCSK9-mC before bleach for which fluorescence recovery after photobleaching (FRAP) values were obtained (FRAP). Inverse FRAP (iFRAP) studies in the presence of 200 μg/ml cycloheximide to block protein synthesis (as described in Materials and Methods) were performed for corresponding PCSK9-mC shown in (A). For each PCSK9-mC constructs, mobile fraction (%) at the TGN from FRAP and iFRAP were calculated as described in Materials and Methods. Data are representative of at least three independent experiments are shown as the mean ± S.E.M. Statistical significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Scale bars , 10 μm.

    Journal: PLoS ONE

    Article Title: Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain

    doi: 10.1371/journal.pone.0157230

    Figure Lengend Snippet: Effect of PCSK9 natural mutants on TGN localization and trafficking dynamics by FRAP and iFRAP. (A) HepG2 cells were transfected with PCSK9-mC WT, GOF S127R, D129G, D374Y or LOF R46L mutants (red) and TGN localization was determined by colocalization (arrows) with Golgin-97 (green) and analysis by confocal microscopy. Nuclei were labeled with TO-PRO-3 Iodide (cyan). (B) Half time (T 1/2 ; min) and mobile fraction (%) calculated from FRAP (ER ➜ Golgi) and iFRAP (Golgi ➜ plasma membrane) experiments in living HepG2 cells for corresponding PCSK9-mC constructs shown in (A). Dashed square represent typical TGN localization of PCSK9-mC before bleach for which fluorescence recovery after photobleaching (FRAP) values were obtained (FRAP). Inverse FRAP (iFRAP) studies in the presence of 200 μg/ml cycloheximide to block protein synthesis (as described in Materials and Methods) were performed for corresponding PCSK9-mC shown in (A). For each PCSK9-mC constructs, mobile fraction (%) at the TGN from FRAP and iFRAP were calculated as described in Materials and Methods. Data are representative of at least three independent experiments are shown as the mean ± S.E.M. Statistical significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Scale bars , 10 μm.

    Article Snippet: The cells were then incubated for 30 min with 1% BSA (Fraction V; Cat. #BP1605, Sigma) containing or not 0.1% Triton X-100, followed by overnight incubation at 4°C with rabbit anti-human PCSK9 (1:250), mouse anti-V5 (1/500; Cat. #R960-025, Life Technologies) and mouse anti-Golgin-97 (1/500; Cat. #sc-59820, Santa Cruz Biotechnology).

    Techniques: Transfection, Confocal Microscopy, Labeling, Construct, Fluorescence, Blocking Assay

    Deletion of PCSK9 hinge region does not affect CTD localization at the TGN. PCSK9 molecular structure was extracted from PDB 2P4E using MacPymol software. Prodomain (PRO), catalytic domain (CAT) and its LDLR-interacting residues (in pink), hinge region in blue and position R434 in red (site of LOF R434W) and C-terminal domain (CTD) are indicated. HepG2 cells were transfected with V5-tagged ΔHinge-CTD (Δaa 422-439-CTD), immunolabeled with the anti-V5 and anti-Golgin-97 antibodies and their localization was analyzed by confocal microscopy ( lower panels ). Colocalization was quantified using Pearson’s correlation coefficient (Rcoloc) (minimum of 10 cells were analyzed). Data are representative of at least three independent experiments. Scale bar , 10 μm.

    Journal: PLoS ONE

    Article Title: Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain

    doi: 10.1371/journal.pone.0157230

    Figure Lengend Snippet: Deletion of PCSK9 hinge region does not affect CTD localization at the TGN. PCSK9 molecular structure was extracted from PDB 2P4E using MacPymol software. Prodomain (PRO), catalytic domain (CAT) and its LDLR-interacting residues (in pink), hinge region in blue and position R434 in red (site of LOF R434W) and C-terminal domain (CTD) are indicated. HepG2 cells were transfected with V5-tagged ΔHinge-CTD (Δaa 422-439-CTD), immunolabeled with the anti-V5 and anti-Golgin-97 antibodies and their localization was analyzed by confocal microscopy ( lower panels ). Colocalization was quantified using Pearson’s correlation coefficient (Rcoloc) (minimum of 10 cells were analyzed). Data are representative of at least three independent experiments. Scale bar , 10 μm.

    Article Snippet: The cells were then incubated for 30 min with 1% BSA (Fraction V; Cat. #BP1605, Sigma) containing or not 0.1% Triton X-100, followed by overnight incubation at 4°C with rabbit anti-human PCSK9 (1:250), mouse anti-V5 (1/500; Cat. #R960-025, Life Technologies) and mouse anti-Golgin-97 (1/500; Cat. #sc-59820, Santa Cruz Biotechnology).

    Techniques: Software, Transfection, Immunolabeling, Confocal Microscopy

    PCSK9 localizes at the TGN via its CTD. (A) Ultrastructural localization of PCSK9 in normal human liver tissue sections shows the immunogold labeling over the rough endoplasmic reticulum (RER; left panel), Golgi apparatus (G), mulitvesicular bodies (MVB) and late endosomes (LE) (middle and left panels) of hepatocytes. Arrowheads denotes inward budding vesicles that are characteristic of MVBs (right panel). Note the presence of very few gold particles over mitochondria (M) and glycogen (Gly) indicating the specificity of the labeling. Omission of the primary antibody resulted in the absence of specific labeling (control experiment, Ctl; inset). Magnification X 25000. (B) Co-localization of endogenous PCSK9 with the TGN marker Golgin-97 as visualized by confocal microscopy ( left panels , white arrows ). Specificity of PCSK9 immunolabeling was tested following overnight pre-incubation of the anti-PCSK9 Ab with 1 μg/ml recombinant human PCSK9 ( right panels , immunoadsorption ). (C) HepG2 cells were transfected with V5-tagged FL PCSK9, CTD, ΔCTD or LOF R434W mutant and co-localization with Golgin-97 was analyzed by confocal microscopy. (B, C) Colocalization was quantified using Pearson’s correlation coefficient (Rcoloc) (minimum of 10 cells were analyzed). Data are representative of at least three independent experiments. Scale bars , 20 μm.

    Journal: PLoS ONE

    Article Title: Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain

    doi: 10.1371/journal.pone.0157230

    Figure Lengend Snippet: PCSK9 localizes at the TGN via its CTD. (A) Ultrastructural localization of PCSK9 in normal human liver tissue sections shows the immunogold labeling over the rough endoplasmic reticulum (RER; left panel), Golgi apparatus (G), mulitvesicular bodies (MVB) and late endosomes (LE) (middle and left panels) of hepatocytes. Arrowheads denotes inward budding vesicles that are characteristic of MVBs (right panel). Note the presence of very few gold particles over mitochondria (M) and glycogen (Gly) indicating the specificity of the labeling. Omission of the primary antibody resulted in the absence of specific labeling (control experiment, Ctl; inset). Magnification X 25000. (B) Co-localization of endogenous PCSK9 with the TGN marker Golgin-97 as visualized by confocal microscopy ( left panels , white arrows ). Specificity of PCSK9 immunolabeling was tested following overnight pre-incubation of the anti-PCSK9 Ab with 1 μg/ml recombinant human PCSK9 ( right panels , immunoadsorption ). (C) HepG2 cells were transfected with V5-tagged FL PCSK9, CTD, ΔCTD or LOF R434W mutant and co-localization with Golgin-97 was analyzed by confocal microscopy. (B, C) Colocalization was quantified using Pearson’s correlation coefficient (Rcoloc) (minimum of 10 cells were analyzed). Data are representative of at least three independent experiments. Scale bars , 20 μm.

    Article Snippet: The cells were then incubated for 30 min with 1% BSA (Fraction V; Cat. #BP1605, Sigma) containing or not 0.1% Triton X-100, followed by overnight incubation at 4°C with rabbit anti-human PCSK9 (1:250), mouse anti-V5 (1/500; Cat. #R960-025, Life Technologies) and mouse anti-Golgin-97 (1/500; Cat. #sc-59820, Santa Cruz Biotechnology).

    Techniques: Labeling, CTL Assay, Marker, Confocal Microscopy, Immunolabeling, Incubation, Recombinant, Transfection, Mutagenesis

    Fusion of monomeric fluorescent proteins to PCSK9 and LDLR. (A) HEK293 cells were transiently transfected with plasmids encoding WT PCSK9-mCherry (mC) or its GOF D374Y mutant and expression was analyzed by fluorescence microscopy with a 20X objective. (B) HEK293 cells were transfected without (Empty) or with WT LDLR or its EGF-A D331E mutant, incubated for 24 h (left panels) or 4 h (right panels) in DMEM or with conditioned media from PCSK9-mC transfected HEK293 cells. LDLR was revealed under non-permeabilizing conditions and protein localization was analyzed by confocal immunofluorescence microscopy. Magnified images of area within dashed lines are shown (bottom panels). Scale bars ; 20 μm. (C) Immunoblots (IB) of conditioned media of PCSK9 natural mutants and truncation variants obtained from transiently transfected HEK293 cells ( input media ). Corresponding media were incubated overnight on HepG2 cells and LDLR and β-actin (loading control) protein levels were analyzed by IB. (D) HEK293 cells were transfected with LDLR-EGFP without (Empty) or with PCSK9 D374Y-mC and both proteins were analyzed by confocal microscopy ( left panels ) and IB ( right panels ). Scale bar = 20 μM. Data are representative of at least three independent experiments.

    Journal: PLoS ONE

    Article Title: Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain

    doi: 10.1371/journal.pone.0157230

    Figure Lengend Snippet: Fusion of monomeric fluorescent proteins to PCSK9 and LDLR. (A) HEK293 cells were transiently transfected with plasmids encoding WT PCSK9-mCherry (mC) or its GOF D374Y mutant and expression was analyzed by fluorescence microscopy with a 20X objective. (B) HEK293 cells were transfected without (Empty) or with WT LDLR or its EGF-A D331E mutant, incubated for 24 h (left panels) or 4 h (right panels) in DMEM or with conditioned media from PCSK9-mC transfected HEK293 cells. LDLR was revealed under non-permeabilizing conditions and protein localization was analyzed by confocal immunofluorescence microscopy. Magnified images of area within dashed lines are shown (bottom panels). Scale bars ; 20 μm. (C) Immunoblots (IB) of conditioned media of PCSK9 natural mutants and truncation variants obtained from transiently transfected HEK293 cells ( input media ). Corresponding media were incubated overnight on HepG2 cells and LDLR and β-actin (loading control) protein levels were analyzed by IB. (D) HEK293 cells were transfected with LDLR-EGFP without (Empty) or with PCSK9 D374Y-mC and both proteins were analyzed by confocal microscopy ( left panels ) and IB ( right panels ). Scale bar = 20 μM. Data are representative of at least three independent experiments.

    Article Snippet: The cells were then incubated for 30 min with 1% BSA (Fraction V; Cat. #BP1605, Sigma) containing or not 0.1% Triton X-100, followed by overnight incubation at 4°C with rabbit anti-human PCSK9 (1:250), mouse anti-V5 (1/500; Cat. #R960-025, Life Technologies) and mouse anti-Golgin-97 (1/500; Cat. #sc-59820, Santa Cruz Biotechnology).

    Techniques: Transfection, Mutagenesis, Expressing, Fluorescence, Microscopy, Incubation, Immunofluorescence, Western Blot, Confocal Microscopy

    Dual fluorescence cell-based assay using PCSK9-mC and LDLR-EGFP. Schematic representation of different readouts that could be obtained from PCSK9-mC and LDLR-EGFP co-expressing cells is shown ( upper panels ). HEK293 cells were transfected with LDLR-EGFP and incubated for 4 h with WT PCSK9-mC or PCSK9 D374Y-mC containing media obtained from transfected cells without or with 4 nM anti-PCSK9 neutralizing antibody pre-incubated overnight (lower panels). Selected regions (dashed squares) were digitally zoomed 5X (magnified images, MAG). Scale bars = 20 μM. Data are representative of at least three independent experiments.

    Journal: PLoS ONE

    Article Title: Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain

    doi: 10.1371/journal.pone.0157230

    Figure Lengend Snippet: Dual fluorescence cell-based assay using PCSK9-mC and LDLR-EGFP. Schematic representation of different readouts that could be obtained from PCSK9-mC and LDLR-EGFP co-expressing cells is shown ( upper panels ). HEK293 cells were transfected with LDLR-EGFP and incubated for 4 h with WT PCSK9-mC or PCSK9 D374Y-mC containing media obtained from transfected cells without or with 4 nM anti-PCSK9 neutralizing antibody pre-incubated overnight (lower panels). Selected regions (dashed squares) were digitally zoomed 5X (magnified images, MAG). Scale bars = 20 μM. Data are representative of at least three independent experiments.

    Article Snippet: The cells were then incubated for 30 min with 1% BSA (Fraction V; Cat. #BP1605, Sigma) containing or not 0.1% Triton X-100, followed by overnight incubation at 4°C with rabbit anti-human PCSK9 (1:250), mouse anti-V5 (1/500; Cat. #R960-025, Life Technologies) and mouse anti-Golgin-97 (1/500; Cat. #sc-59820, Santa Cruz Biotechnology).

    Techniques: Fluorescence, Cell Based Assay, Expressing, Transfection, Incubation

    PCSK9 CTD is required for efficient LDLR-mediated endocytosis of PCSK9. (A) HepG2 cells transfected with LDLR-EGFP were incubated 5h with conditioned media obtained from HEK293 cells transected without (Empty) or with PCSK9 WT-, D374Y-, ΔCTD- or CTD-mC. PCSK9-mC constructs and LDLR-EGFP were analyzed by live-cell confocal microscopy. Selected regions (dashed squares) were digitally zoomed 5X (MAG). Scale bars = 20 μM. (B) Quantification of the number of intracellular red fluorescent puncta per cell expressing LDLR-EGFP for each PCSK9 mCherry fusion construct described in (A) after incubation for 0, 60, 120, 180, 240 and 300 min. PCSK9 WT-mC (n = 37 cells), D374Y-mC (n = 62 cells), ΔCTD-mC (n = 31 cells) or CTD-mC (n = 52 cells). (C) HEK293 cells were transfected without (-) or with WT LDLR or its EGF-A D331E mutant and incubated for 6 h with DMEM alone (-) or with normalized conditioned media obtained from FL or ΔCTD PCSK9-mC transfected cells (input media). Cell-associated PCSK9-mC, LDLR and β-actin (loading control) protein levels were analyzed by IB. Data are representative of at least three independent experiments.

    Journal: PLoS ONE

    Article Title: Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain

    doi: 10.1371/journal.pone.0157230

    Figure Lengend Snippet: PCSK9 CTD is required for efficient LDLR-mediated endocytosis of PCSK9. (A) HepG2 cells transfected with LDLR-EGFP were incubated 5h with conditioned media obtained from HEK293 cells transected without (Empty) or with PCSK9 WT-, D374Y-, ΔCTD- or CTD-mC. PCSK9-mC constructs and LDLR-EGFP were analyzed by live-cell confocal microscopy. Selected regions (dashed squares) were digitally zoomed 5X (MAG). Scale bars = 20 μM. (B) Quantification of the number of intracellular red fluorescent puncta per cell expressing LDLR-EGFP for each PCSK9 mCherry fusion construct described in (A) after incubation for 0, 60, 120, 180, 240 and 300 min. PCSK9 WT-mC (n = 37 cells), D374Y-mC (n = 62 cells), ΔCTD-mC (n = 31 cells) or CTD-mC (n = 52 cells). (C) HEK293 cells were transfected without (-) or with WT LDLR or its EGF-A D331E mutant and incubated for 6 h with DMEM alone (-) or with normalized conditioned media obtained from FL or ΔCTD PCSK9-mC transfected cells (input media). Cell-associated PCSK9-mC, LDLR and β-actin (loading control) protein levels were analyzed by IB. Data are representative of at least three independent experiments.

    Article Snippet: The cells were then incubated for 30 min with 1% BSA (Fraction V; Cat. #BP1605, Sigma) containing or not 0.1% Triton X-100, followed by overnight incubation at 4°C with rabbit anti-human PCSK9 (1:250), mouse anti-V5 (1/500; Cat. #R960-025, Life Technologies) and mouse anti-Golgin-97 (1/500; Cat. #sc-59820, Santa Cruz Biotechnology).

    Techniques: Transfection, Incubation, Construct, Confocal Microscopy, Expressing, Mutagenesis

    SR-SIM reveals that f-actin and drebrin form a cortical collar around microtubules in the proximal leading process. ( a , b ) SR-SIM imaging of CGNs with dilated proximal leading processes expressing ( a ) GPI-pHluorin (green), drebrin E 2x-KO1 (red) and SiR-tubulin (blue; n =21 cells analysed) or ( b ) EGFP-UTRCH (green), Drebrin E 2x-KO1 (red) and SiR-tubulin (blue; n =18 cells analysed). Scale bar, 1 μm ( a ). The RGB plot to the right of each representative image shows the well-resolved Gaussian FWHM peaks detected for the line scan in each image (dashed white line). The box plots at far right show the measured distances between the centroid positions of the Gaussian peak for each fluorescent probe. Whiskers on the box plot show maximum and minimum data points, box borders show first and third quartiles and the line in the box show the median. ( c ) Drebrin is an f-actin and +TIP binding protein. Consistent with this interaction, LLS microscopy shows that microtubule +TIPs (labelled with EB3-2x Venus, green) pass through the drebrin E-labelled domain (labelled with drebrin E 2x-KO1, red) in the proximal leading process. Scale bar, 2μm ( c ). Time stamp=min:sec.

    Journal: Nature Communications

    Article Title: Drebrin-mediated microtubule–actomyosin coupling steers cerebellar granule neuron nucleokinesis and migration pathway selection

    doi: 10.1038/ncomms14484

    Figure Lengend Snippet: SR-SIM reveals that f-actin and drebrin form a cortical collar around microtubules in the proximal leading process. ( a , b ) SR-SIM imaging of CGNs with dilated proximal leading processes expressing ( a ) GPI-pHluorin (green), drebrin E 2x-KO1 (red) and SiR-tubulin (blue; n =21 cells analysed) or ( b ) EGFP-UTRCH (green), Drebrin E 2x-KO1 (red) and SiR-tubulin (blue; n =18 cells analysed). Scale bar, 1 μm ( a ). The RGB plot to the right of each representative image shows the well-resolved Gaussian FWHM peaks detected for the line scan in each image (dashed white line). The box plots at far right show the measured distances between the centroid positions of the Gaussian peak for each fluorescent probe. Whiskers on the box plot show maximum and minimum data points, box borders show first and third quartiles and the line in the box show the median. ( c ) Drebrin is an f-actin and +TIP binding protein. Consistent with this interaction, LLS microscopy shows that microtubule +TIPs (labelled with EB3-2x Venus, green) pass through the drebrin E-labelled domain (labelled with drebrin E 2x-KO1, red) in the proximal leading process. Scale bar, 2μm ( c ). Time stamp=min:sec.

    Article Snippet: Antibodies used were mouse α-drebrin A/E (Clone 2E11, Sigma Catalogue Number SAB1402168, 1:5,000 dil), rabbit α-drebrin A/E (Abcam Catalogue Number ab11068, 1:5,000 dil), rabbit drebrin A (DAS2, Immuno-Biological Laboratories Co., Ltd, Catalogue Number #28023, 1:100 dil), rabbit α-myosin IIB (Covance Catalogue Number 909901, 1:100 dil), mouse α-alpha tubulin (Tub2.1, Sigma Catalogue Number T4026, 1:1,000 dil), and α-phospho drebrin Ser142 (clone 3C14, EMD Milipore Catalogue Number MABN833, 1:100 dil).

    Techniques: Imaging, Expressing, Binding Assay, Microscopy, Size-exclusion Chromatography

    Siah2 antagonizes drebrin function. ( a ) CGNs in culture expressed Emerald MAPT and RFP LIFEACT to label the microtubule and actin cytoskeleton. Time-lapse imaging shows that Siah2-insensitive drebrin NxN rescues a Siah2 gain-of-function phenotype. Top row: Control CGNs have long neurites. Middle row: Siah2 gain of function inhibits CGN neurite extension, induces a radial microtubule cytoskeleton and locks CGNs in a mesenchymal morphology. Bottom row: CGNs expressing drebrin NxN and Siah2 have neurites and microtubule cytoskeleton similar to controls. Scale bar, 10 μm. ( b ) Quantitation of imaging sequences shown in a ( n ≥337 cells analysed for each condition, P

    Journal: Nature Communications

    Article Title: Drebrin-mediated microtubule–actomyosin coupling steers cerebellar granule neuron nucleokinesis and migration pathway selection

    doi: 10.1038/ncomms14484

    Figure Lengend Snippet: Siah2 antagonizes drebrin function. ( a ) CGNs in culture expressed Emerald MAPT and RFP LIFEACT to label the microtubule and actin cytoskeleton. Time-lapse imaging shows that Siah2-insensitive drebrin NxN rescues a Siah2 gain-of-function phenotype. Top row: Control CGNs have long neurites. Middle row: Siah2 gain of function inhibits CGN neurite extension, induces a radial microtubule cytoskeleton and locks CGNs in a mesenchymal morphology. Bottom row: CGNs expressing drebrin NxN and Siah2 have neurites and microtubule cytoskeleton similar to controls. Scale bar, 10 μm. ( b ) Quantitation of imaging sequences shown in a ( n ≥337 cells analysed for each condition, P

    Article Snippet: Antibodies used were mouse α-drebrin A/E (Clone 2E11, Sigma Catalogue Number SAB1402168, 1:5,000 dil), rabbit α-drebrin A/E (Abcam Catalogue Number ab11068, 1:5,000 dil), rabbit drebrin A (DAS2, Immuno-Biological Laboratories Co., Ltd, Catalogue Number #28023, 1:100 dil), rabbit α-myosin IIB (Covance Catalogue Number 909901, 1:100 dil), mouse α-alpha tubulin (Tub2.1, Sigma Catalogue Number T4026, 1:1,000 dil), and α-phospho drebrin Ser142 (clone 3C14, EMD Milipore Catalogue Number MABN833, 1:100 dil).

    Techniques: Imaging, Expressing, Quantitation Assay

    LLS microscopy reveals unappreciated relationships between cytoskeleton and plasma membrane in the proximal leading process. CGNs cultured in conditions that recapitulate radial migration were electroporated with expression vectors encoding the indicated fluorescently labelled live-cell imaging probes. The displayed images are maximum intensity projections of selected time points of cultures imaged via LLS microscopy 18–24 h post transfection. Scale bar, 2 μm. ( a ) Simultaneous imaging of plasma membrane (GPI-pHluorin, green) and drebrin (labelled with drebrin E 2x-KO1, red) reveals that drebrin is submembranous during its anterograde translocation. ( b ) f-Actin (labelled with EGFP-UTRCH, green) accumulates in the leading process before somal translocation. Initially, drebrin (labelled with drebrin E 2x-KO1, red) is localized mostly in the cell body but becomes restricted to the leading process f-actin domain. ( c ) Debrin (labelled with drebrin E 2x-KO1, red) accumulates around microtubules located in a well-dilated proximal leading process (labelled with MAPT-Emerald, green) before somal translocation. Note that drebrin appears to be more cortically restricted than the microtubules. ( d ) Simultaneous imaging of JAM-C adhesions (JAM-C-pHluorin, green) and drebrin (labelled with drebrin E 2x-KO1, red) reveals that a subset of JAM-C adhesions are located in the drebrin contractile domain. Scale bar, 2 μm ( a ). Time stamp=min:sec.

    Journal: Nature Communications

    Article Title: Drebrin-mediated microtubule–actomyosin coupling steers cerebellar granule neuron nucleokinesis and migration pathway selection

    doi: 10.1038/ncomms14484

    Figure Lengend Snippet: LLS microscopy reveals unappreciated relationships between cytoskeleton and plasma membrane in the proximal leading process. CGNs cultured in conditions that recapitulate radial migration were electroporated with expression vectors encoding the indicated fluorescently labelled live-cell imaging probes. The displayed images are maximum intensity projections of selected time points of cultures imaged via LLS microscopy 18–24 h post transfection. Scale bar, 2 μm. ( a ) Simultaneous imaging of plasma membrane (GPI-pHluorin, green) and drebrin (labelled with drebrin E 2x-KO1, red) reveals that drebrin is submembranous during its anterograde translocation. ( b ) f-Actin (labelled with EGFP-UTRCH, green) accumulates in the leading process before somal translocation. Initially, drebrin (labelled with drebrin E 2x-KO1, red) is localized mostly in the cell body but becomes restricted to the leading process f-actin domain. ( c ) Debrin (labelled with drebrin E 2x-KO1, red) accumulates around microtubules located in a well-dilated proximal leading process (labelled with MAPT-Emerald, green) before somal translocation. Note that drebrin appears to be more cortically restricted than the microtubules. ( d ) Simultaneous imaging of JAM-C adhesions (JAM-C-pHluorin, green) and drebrin (labelled with drebrin E 2x-KO1, red) reveals that a subset of JAM-C adhesions are located in the drebrin contractile domain. Scale bar, 2 μm ( a ). Time stamp=min:sec.

    Article Snippet: Antibodies used were mouse α-drebrin A/E (Clone 2E11, Sigma Catalogue Number SAB1402168, 1:5,000 dil), rabbit α-drebrin A/E (Abcam Catalogue Number ab11068, 1:5,000 dil), rabbit drebrin A (DAS2, Immuno-Biological Laboratories Co., Ltd, Catalogue Number #28023, 1:100 dil), rabbit α-myosin IIB (Covance Catalogue Number 909901, 1:100 dil), mouse α-alpha tubulin (Tub2.1, Sigma Catalogue Number T4026, 1:1,000 dil), and α-phospho drebrin Ser142 (clone 3C14, EMD Milipore Catalogue Number MABN833, 1:100 dil).

    Techniques: Microscopy, Cell Culture, Migration, Expressing, Live Cell Imaging, Transfection, Imaging, Translocation Assay, Size-exclusion Chromatography

    Drebrin function is required for directed movement of two-stroke motility in vitro . CGNs were transfected with expression vectors encoding Centrin2-Venus, H2B-mCherry and the indicated experimental constructs. Time-lapse imaging was used to monitor two-stroke nucleokinesis in migrating CGNs in which drebrin was silenced ( a , b ) ( n ≥57 cells analysed for each condition and organelle) or when the dominant-negative EB3M was overexpressed ( c , d ) ( n ≥70 cells analysed for each condition and organelle). The multicolour images in a , c show nuclear positions for selected time points from representative imaging sequences, whereas b , d show centrosome positions (see the colour key for time points). A polar efficiency and an MSD plot are provided for each organelle. Drebrin loss of function and dominant-negative inhibition of plus-end binding is accompanied by less efficient somal and centrosome motility relative to calculated FDVs. Scale bars, 10μm. Error bars show ±s.d.

    Journal: Nature Communications

    Article Title: Drebrin-mediated microtubule–actomyosin coupling steers cerebellar granule neuron nucleokinesis and migration pathway selection

    doi: 10.1038/ncomms14484

    Figure Lengend Snippet: Drebrin function is required for directed movement of two-stroke motility in vitro . CGNs were transfected with expression vectors encoding Centrin2-Venus, H2B-mCherry and the indicated experimental constructs. Time-lapse imaging was used to monitor two-stroke nucleokinesis in migrating CGNs in which drebrin was silenced ( a , b ) ( n ≥57 cells analysed for each condition and organelle) or when the dominant-negative EB3M was overexpressed ( c , d ) ( n ≥70 cells analysed for each condition and organelle). The multicolour images in a , c show nuclear positions for selected time points from representative imaging sequences, whereas b , d show centrosome positions (see the colour key for time points). A polar efficiency and an MSD plot are provided for each organelle. Drebrin loss of function and dominant-negative inhibition of plus-end binding is accompanied by less efficient somal and centrosome motility relative to calculated FDVs. Scale bars, 10μm. Error bars show ±s.d.

    Article Snippet: Antibodies used were mouse α-drebrin A/E (Clone 2E11, Sigma Catalogue Number SAB1402168, 1:5,000 dil), rabbit α-drebrin A/E (Abcam Catalogue Number ab11068, 1:5,000 dil), rabbit drebrin A (DAS2, Immuno-Biological Laboratories Co., Ltd, Catalogue Number #28023, 1:100 dil), rabbit α-myosin IIB (Covance Catalogue Number 909901, 1:100 dil), mouse α-alpha tubulin (Tub2.1, Sigma Catalogue Number T4026, 1:1,000 dil), and α-phospho drebrin Ser142 (clone 3C14, EMD Milipore Catalogue Number MABN833, 1:100 dil).

    Techniques: In Vitro, Transfection, Expressing, Construct, Imaging, Dominant Negative Mutation, Inhibition, Binding Assay

    Ex vivo analysis of drebrin loss of function. ( a ) Cerebella from P7 mice were electroporated, and slices grown in ex vivo culture for 48 h. CGNs were electroporated with a vector encoding H2B-mCherry either alone or in combination with the indicated expression vectors. EB1M and EB3M expression was driven in GNPs using pMath1 and in CGNs using pNeuroD. Each representative image is oriented with the cerebellar slice surface to the left, and the presence of red nuclei in the centre or right of the image indicates cells that have left the GZ. The histograms below each representative image show the binned migration distance distribution for each condition ( n ≥2,500 cells analysed for each condition, χ 2 test P value

    Journal: Nature Communications

    Article Title: Drebrin-mediated microtubule–actomyosin coupling steers cerebellar granule neuron nucleokinesis and migration pathway selection

    doi: 10.1038/ncomms14484

    Figure Lengend Snippet: Ex vivo analysis of drebrin loss of function. ( a ) Cerebella from P7 mice were electroporated, and slices grown in ex vivo culture for 48 h. CGNs were electroporated with a vector encoding H2B-mCherry either alone or in combination with the indicated expression vectors. EB1M and EB3M expression was driven in GNPs using pMath1 and in CGNs using pNeuroD. Each representative image is oriented with the cerebellar slice surface to the left, and the presence of red nuclei in the centre or right of the image indicates cells that have left the GZ. The histograms below each representative image show the binned migration distance distribution for each condition ( n ≥2,500 cells analysed for each condition, χ 2 test P value

    Article Snippet: Antibodies used were mouse α-drebrin A/E (Clone 2E11, Sigma Catalogue Number SAB1402168, 1:5,000 dil), rabbit α-drebrin A/E (Abcam Catalogue Number ab11068, 1:5,000 dil), rabbit drebrin A (DAS2, Immuno-Biological Laboratories Co., Ltd, Catalogue Number #28023, 1:100 dil), rabbit α-myosin IIB (Covance Catalogue Number 909901, 1:100 dil), mouse α-alpha tubulin (Tub2.1, Sigma Catalogue Number T4026, 1:1,000 dil), and α-phospho drebrin Ser142 (clone 3C14, EMD Milipore Catalogue Number MABN833, 1:100 dil).

    Techniques: Ex Vivo, Mouse Assay, Plasmid Preparation, Expressing, Migration

    Drebrin–microtubule interactions are required for proximal leading process microtubule movement in vitro . CGNs were transfected with expression vectors encoding photoactivatable EGFP-α-tubulin (green), RFP-UTRCH-ABD (f-actin label, red), or the indicated EB3M and drebrin shRNA constructs. ( a ) Schematic of the photoactivation strategy: a 405-nm laser was used to activate a microtubule fiduciary mark within well-dilated CGN proximal leading processes, and time-lapse imaging was used to monitor fiduciary mark movement during two-stroke nucleokinesis. Movements with positive values were towards the distal tip of the leading process (that is, in the direction of migration), whereas negative movement values were towards the cell body. ( b ) Box plot showing the average advance of the fiduciary mark in control, EB3M-expressing or drebrin -silenced CGNs. Control marks moved in the direction of migration, whereas the marks in EB3M-expressing cells retreated towards the soma ( n =15 for control, 27 for EB3M-expressing and 15 for drebrin- silenced cells, P

    Journal: Nature Communications

    Article Title: Drebrin-mediated microtubule–actomyosin coupling steers cerebellar granule neuron nucleokinesis and migration pathway selection

    doi: 10.1038/ncomms14484

    Figure Lengend Snippet: Drebrin–microtubule interactions are required for proximal leading process microtubule movement in vitro . CGNs were transfected with expression vectors encoding photoactivatable EGFP-α-tubulin (green), RFP-UTRCH-ABD (f-actin label, red), or the indicated EB3M and drebrin shRNA constructs. ( a ) Schematic of the photoactivation strategy: a 405-nm laser was used to activate a microtubule fiduciary mark within well-dilated CGN proximal leading processes, and time-lapse imaging was used to monitor fiduciary mark movement during two-stroke nucleokinesis. Movements with positive values were towards the distal tip of the leading process (that is, in the direction of migration), whereas negative movement values were towards the cell body. ( b ) Box plot showing the average advance of the fiduciary mark in control, EB3M-expressing or drebrin -silenced CGNs. Control marks moved in the direction of migration, whereas the marks in EB3M-expressing cells retreated towards the soma ( n =15 for control, 27 for EB3M-expressing and 15 for drebrin- silenced cells, P

    Article Snippet: Antibodies used were mouse α-drebrin A/E (Clone 2E11, Sigma Catalogue Number SAB1402168, 1:5,000 dil), rabbit α-drebrin A/E (Abcam Catalogue Number ab11068, 1:5,000 dil), rabbit drebrin A (DAS2, Immuno-Biological Laboratories Co., Ltd, Catalogue Number #28023, 1:100 dil), rabbit α-myosin IIB (Covance Catalogue Number 909901, 1:100 dil), mouse α-alpha tubulin (Tub2.1, Sigma Catalogue Number T4026, 1:1,000 dil), and α-phospho drebrin Ser142 (clone 3C14, EMD Milipore Catalogue Number MABN833, 1:100 dil).

    Techniques: In Vitro, Transfection, Expressing, shRNA, Construct, Imaging, Migration

    Drebrin protein is expressed in differentiated CGNs and is dynamically localized to the leading process during two-stroke motility. ( a – c ) Immunohistochemical examination of drebrin expression in the P7 mouse cerebellum. In the P7 cerebellum, drebrin (red) is complementarily expressed with the GNP markers Ki67 (green) ( a ) or Zeb1 (green) ( b ) and is co-expressed in differentiated CGNs with p27Kip (green) ( c ), scale bar for each image equals 50 μm. Drebrin A expression (red) is minimal at P7 ( d ), scale bar, 10 μm. ( e – g ) Immunocytochemical examination of drebrin expression in CGNs grown in culture. Expression of drebrin protein (green) is coincident with myosin IIB, alpha-tubulin, and drebrin phospho-Ser142 immunoreactivity (all red) (lp=leading process), scale bar for each equals 5 μm. ( h ) Drebrin (labelled with drebrin E 2x-KO1, red) is initially localized in the cell body but becomes restricted to the leading process f-actin domain, where it appears to contract around the time of somal translocation. Around the time of local contraction, bending is observed in the microtubule cytoskeleton (labelled by YFP-Map2C, white arrows). Scale bar, 10 μm. Time stamp=hours:min:sec. ( i ) Adaptive volumetric kymographs of the sequences shown in h : the leading process and direction of migration is towards the right of the panel (dashed blue line=somal boundaries; solid blue line=soma centre; red=drebrin E 2x-KO1; green=YFP-Map2C). The yellow box highlights the time-lapse frames shown in h , in which a subpopulation of drebrin flows down the leading process in the direction of migration before the contraction of the leading process drebrin domain. Arrows indicate the borders of the drebrin domain as it contracts during a migration cycle. ( j ) Analysis of the percentage of regional drebrin localization signal in the soma and leading or trailing process in migrating neurons expressing drebrin E 2x-KO1, in which drebrin accumulates in the leading process in the active phase of migration.

    Journal: Nature Communications

    Article Title: Drebrin-mediated microtubule–actomyosin coupling steers cerebellar granule neuron nucleokinesis and migration pathway selection

    doi: 10.1038/ncomms14484

    Figure Lengend Snippet: Drebrin protein is expressed in differentiated CGNs and is dynamically localized to the leading process during two-stroke motility. ( a – c ) Immunohistochemical examination of drebrin expression in the P7 mouse cerebellum. In the P7 cerebellum, drebrin (red) is complementarily expressed with the GNP markers Ki67 (green) ( a ) or Zeb1 (green) ( b ) and is co-expressed in differentiated CGNs with p27Kip (green) ( c ), scale bar for each image equals 50 μm. Drebrin A expression (red) is minimal at P7 ( d ), scale bar, 10 μm. ( e – g ) Immunocytochemical examination of drebrin expression in CGNs grown in culture. Expression of drebrin protein (green) is coincident with myosin IIB, alpha-tubulin, and drebrin phospho-Ser142 immunoreactivity (all red) (lp=leading process), scale bar for each equals 5 μm. ( h ) Drebrin (labelled with drebrin E 2x-KO1, red) is initially localized in the cell body but becomes restricted to the leading process f-actin domain, where it appears to contract around the time of somal translocation. Around the time of local contraction, bending is observed in the microtubule cytoskeleton (labelled by YFP-Map2C, white arrows). Scale bar, 10 μm. Time stamp=hours:min:sec. ( i ) Adaptive volumetric kymographs of the sequences shown in h : the leading process and direction of migration is towards the right of the panel (dashed blue line=somal boundaries; solid blue line=soma centre; red=drebrin E 2x-KO1; green=YFP-Map2C). The yellow box highlights the time-lapse frames shown in h , in which a subpopulation of drebrin flows down the leading process in the direction of migration before the contraction of the leading process drebrin domain. Arrows indicate the borders of the drebrin domain as it contracts during a migration cycle. ( j ) Analysis of the percentage of regional drebrin localization signal in the soma and leading or trailing process in migrating neurons expressing drebrin E 2x-KO1, in which drebrin accumulates in the leading process in the active phase of migration.

    Article Snippet: Antibodies used were mouse α-drebrin A/E (Clone 2E11, Sigma Catalogue Number SAB1402168, 1:5,000 dil), rabbit α-drebrin A/E (Abcam Catalogue Number ab11068, 1:5,000 dil), rabbit drebrin A (DAS2, Immuno-Biological Laboratories Co., Ltd, Catalogue Number #28023, 1:100 dil), rabbit α-myosin IIB (Covance Catalogue Number 909901, 1:100 dil), mouse α-alpha tubulin (Tub2.1, Sigma Catalogue Number T4026, 1:1,000 dil), and α-phospho drebrin Ser142 (clone 3C14, EMD Milipore Catalogue Number MABN833, 1:100 dil).

    Techniques: Immunohistochemistry, Expressing, Translocation Assay, Size-exclusion Chromatography, Migration

    Siah ubiquitin ligases regulate drebrin protein levels. ( a ) Drebrin domain structure: the C terminus contains two VxP Siah degrons that can be mutated to NxN to inhibit Siah sensitivity. ( b ) Immunohistochemical examination of drebrin expression in the P7 cerebellum. Drebrin (red) expression is low in Siah2-expressing GNPs (green). Scale bar, 50 μm. ( c ) Immunocytochemical examination of drebrin expression in CGN cultures treated with Shh-N-conditioned medium or LacZ-transfected control. Drebrin (red) expression is lower and Siah2 expression higher (green) in Shh-treated cultures, providing a physiological context for drebrin regulation during differentiation. Scale bar, 50 μm. ( d ) CGNs co-nucleofected with drebrin E 2xVenus and Siah2-myc exhibit lower drebrin signal, but not when Siah2-ΔRING is co-expressed. Scale bar, 10 μm. ( e ) Siah2 silencing enhances endogenous drebrin expression in cultured CGNs. CGNs were nucleofected with expression vectors, where a control or Siah2 miR30 shRNA was embedded into the 3′ UTR of a 2xBFP NLS cDNA. After drebrin immunostaining, expression levels were measured and displayed in the accompanying graph. Scale bar, 5 μm (Student's t -test P

    Journal: Nature Communications

    Article Title: Drebrin-mediated microtubule–actomyosin coupling steers cerebellar granule neuron nucleokinesis and migration pathway selection

    doi: 10.1038/ncomms14484

    Figure Lengend Snippet: Siah ubiquitin ligases regulate drebrin protein levels. ( a ) Drebrin domain structure: the C terminus contains two VxP Siah degrons that can be mutated to NxN to inhibit Siah sensitivity. ( b ) Immunohistochemical examination of drebrin expression in the P7 cerebellum. Drebrin (red) expression is low in Siah2-expressing GNPs (green). Scale bar, 50 μm. ( c ) Immunocytochemical examination of drebrin expression in CGN cultures treated with Shh-N-conditioned medium or LacZ-transfected control. Drebrin (red) expression is lower and Siah2 expression higher (green) in Shh-treated cultures, providing a physiological context for drebrin regulation during differentiation. Scale bar, 50 μm. ( d ) CGNs co-nucleofected with drebrin E 2xVenus and Siah2-myc exhibit lower drebrin signal, but not when Siah2-ΔRING is co-expressed. Scale bar, 10 μm. ( e ) Siah2 silencing enhances endogenous drebrin expression in cultured CGNs. CGNs were nucleofected with expression vectors, where a control or Siah2 miR30 shRNA was embedded into the 3′ UTR of a 2xBFP NLS cDNA. After drebrin immunostaining, expression levels were measured and displayed in the accompanying graph. Scale bar, 5 μm (Student's t -test P

    Article Snippet: Antibodies used were mouse α-drebrin A/E (Clone 2E11, Sigma Catalogue Number SAB1402168, 1:5,000 dil), rabbit α-drebrin A/E (Abcam Catalogue Number ab11068, 1:5,000 dil), rabbit drebrin A (DAS2, Immuno-Biological Laboratories Co., Ltd, Catalogue Number #28023, 1:100 dil), rabbit α-myosin IIB (Covance Catalogue Number 909901, 1:100 dil), mouse α-alpha tubulin (Tub2.1, Sigma Catalogue Number T4026, 1:1,000 dil), and α-phospho drebrin Ser142 (clone 3C14, EMD Milipore Catalogue Number MABN833, 1:100 dil).

    Techniques: Immunohistochemistry, Expressing, Transfection, Cell Culture, shRNA, Immunostaining

    RACK1–Lck complexes can form irrespective of Lck kinase activity, but those from activated T-cells contain a sizeable fraction of pY394 Lck . (A) CD4 + T-cells precoated or non-precoated with biotinylated anti-TCR and anti-CD4 mAbs (TCRβ/CD4) were co-aggregated, or not (0 s), with streptavidin for the indicated period of time. RACK1 immunoprecipitates were blotted against pY394 Lck , total Lck, and RACK1. (B,C) Statistical analysis of pY394Lck and total Lck blots from (A) , respectively, represent the normalized to total RACK1 from at least three independent experiments. The statistical analysis presented as mean ± SD was performed using the Student’s two-tailed t -test, * p

    Journal: Frontiers in Immunology

    Article Title: TCR Triggering Induces the Formation of Lck–RACK1–Actinin-1 Multiprotein Network Affecting Lck Redistribution

    doi: 10.3389/fimmu.2016.00449

    Figure Lengend Snippet: RACK1–Lck complexes can form irrespective of Lck kinase activity, but those from activated T-cells contain a sizeable fraction of pY394 Lck . (A) CD4 + T-cells precoated or non-precoated with biotinylated anti-TCR and anti-CD4 mAbs (TCRβ/CD4) were co-aggregated, or not (0 s), with streptavidin for the indicated period of time. RACK1 immunoprecipitates were blotted against pY394 Lck , total Lck, and RACK1. (B,C) Statistical analysis of pY394Lck and total Lck blots from (A) , respectively, represent the normalized to total RACK1 from at least three independent experiments. The statistical analysis presented as mean ± SD was performed using the Student’s two-tailed t -test, * p

    Article Snippet: For western blotting, mouse anti-RACK1, rabbit anti-pY394Lck (Santa Cruz), anti-pY505Lck (Cell signaling), mouse anti-Lck (3A5), and phosphotyrosine-specific platinum 4G10 mAb (Millipore) were used.

    Techniques: Activity Assay, Two Tailed Test

    Adenovirus mediated knockdown of RACK1 impedes translocation of Lck to light DRMs . CD4 + T-cells from TgCAR transgenic mice were infected with either empty control virus (Empty) or with a mixture of adenoviral RACK1-targeting shRNA constructs (shRNA) or were not infected (no virus). (A) The effectiveness of RACK1 downregulation is shown 96 h after infection. Cells were harvested and probed with anti-Lck and anti-RACK1. (B) CD4 + T-cells infected with Empty virus (control) or shRNAs RACK1 virus were activated by TCR/CD4 co-aggregation (30 s), or not (0 s), and the redistribution of Lck to light DRMs was assessed. Raft (#1–3, R) and soluble (#8–10, S) fractions were probed with anti-Lck. Numbers represent relative distribution of Lck to these fractions. Blots are representative of two independent experiments.

    Journal: Frontiers in Immunology

    Article Title: TCR Triggering Induces the Formation of Lck–RACK1–Actinin-1 Multiprotein Network Affecting Lck Redistribution

    doi: 10.3389/fimmu.2016.00449

    Figure Lengend Snippet: Adenovirus mediated knockdown of RACK1 impedes translocation of Lck to light DRMs . CD4 + T-cells from TgCAR transgenic mice were infected with either empty control virus (Empty) or with a mixture of adenoviral RACK1-targeting shRNA constructs (shRNA) or were not infected (no virus). (A) The effectiveness of RACK1 downregulation is shown 96 h after infection. Cells were harvested and probed with anti-Lck and anti-RACK1. (B) CD4 + T-cells infected with Empty virus (control) or shRNAs RACK1 virus were activated by TCR/CD4 co-aggregation (30 s), or not (0 s), and the redistribution of Lck to light DRMs was assessed. Raft (#1–3, R) and soluble (#8–10, S) fractions were probed with anti-Lck. Numbers represent relative distribution of Lck to these fractions. Blots are representative of two independent experiments.

    Article Snippet: For western blotting, mouse anti-RACK1, rabbit anti-pY394Lck (Santa Cruz), anti-pY505Lck (Cell signaling), mouse anti-Lck (3A5), and phosphotyrosine-specific platinum 4G10 mAb (Millipore) were used.

    Techniques: Translocation Assay, Transgenic Assay, Mouse Assay, Infection, shRNA, Construct

    Functional microtubular network is important for RACK1–Lck complex formation upon T-cell activation . (A) CD4 + T-cells were pretreated with latrunculin B (LAT B), nocodazole (NOC), or DMSO (control) and then activated for the indicated period of time followed by lysis in TNE lysis buffer. RACK1 was immunoprecipitated and blotted against Lck and RACK1. Samples from lysates before IP were boiled and subjected to pY western blot analysis with 4G10 antibody. Blots are representative of three independent experiments. (B) Statistical analysis of (A) , Lck panel, represents the relative fold-change of RACK1 co-immunoprecipitated Lck normalized to total RACK1. The untreated control samples were given reference value “1.” The statistical analysis presented as mean ± SD was performed using the Student’s two-tailed t -test, * p

    Journal: Frontiers in Immunology

    Article Title: TCR Triggering Induces the Formation of Lck–RACK1–Actinin-1 Multiprotein Network Affecting Lck Redistribution

    doi: 10.3389/fimmu.2016.00449

    Figure Lengend Snippet: Functional microtubular network is important for RACK1–Lck complex formation upon T-cell activation . (A) CD4 + T-cells were pretreated with latrunculin B (LAT B), nocodazole (NOC), or DMSO (control) and then activated for the indicated period of time followed by lysis in TNE lysis buffer. RACK1 was immunoprecipitated and blotted against Lck and RACK1. Samples from lysates before IP were boiled and subjected to pY western blot analysis with 4G10 antibody. Blots are representative of three independent experiments. (B) Statistical analysis of (A) , Lck panel, represents the relative fold-change of RACK1 co-immunoprecipitated Lck normalized to total RACK1. The untreated control samples were given reference value “1.” The statistical analysis presented as mean ± SD was performed using the Student’s two-tailed t -test, * p

    Article Snippet: For western blotting, mouse anti-RACK1, rabbit anti-pY394Lck (Santa Cruz), anti-pY505Lck (Cell signaling), mouse anti-Lck (3A5), and phosphotyrosine-specific platinum 4G10 mAb (Millipore) were used.

    Techniques: Functional Assay, Activation Assay, Lysis, Immunoprecipitation, Western Blot, Two Tailed Test

    Lck and RACK1 in primary CD4 + T-cell co-redistribute into forming immunological synapse . (A) OVA-pulsed bone marrow-derived dendritic cells (BMDCs) were mixed with CD4 + T-cells from OTII transgenic mice. After 2–3 min, T-cell–APC conjugates were seeded on cover slips, fixed and probed with anti-Lck (red) and anti-RACK1 (green), and visualized by super-resolution N-SIM microscopy. The upper two panels show the fluorescence intensities of individual Lck or RACK1 signals using pseudocolor digital scaling. The panel titled “Detail” shows the magnification of the rectangle inset seen in the Merge image. An image of one representative cell conjugate is presented. Consistent with a previous report ( 52 ), the low expression of Lck was also detected in BMDCs. (B) Statistical analysis of the relative recruitment index (RRI) showed that while both RACK1 and Lck are enriched at the site of the forming IS, RRI for the former is significantly higher than that for Lck ( n = 20), p

    Journal: Frontiers in Immunology

    Article Title: TCR Triggering Induces the Formation of Lck–RACK1–Actinin-1 Multiprotein Network Affecting Lck Redistribution

    doi: 10.3389/fimmu.2016.00449

    Figure Lengend Snippet: Lck and RACK1 in primary CD4 + T-cell co-redistribute into forming immunological synapse . (A) OVA-pulsed bone marrow-derived dendritic cells (BMDCs) were mixed with CD4 + T-cells from OTII transgenic mice. After 2–3 min, T-cell–APC conjugates were seeded on cover slips, fixed and probed with anti-Lck (red) and anti-RACK1 (green), and visualized by super-resolution N-SIM microscopy. The upper two panels show the fluorescence intensities of individual Lck or RACK1 signals using pseudocolor digital scaling. The panel titled “Detail” shows the magnification of the rectangle inset seen in the Merge image. An image of one representative cell conjugate is presented. Consistent with a previous report ( 52 ), the low expression of Lck was also detected in BMDCs. (B) Statistical analysis of the relative recruitment index (RRI) showed that while both RACK1 and Lck are enriched at the site of the forming IS, RRI for the former is significantly higher than that for Lck ( n = 20), p

    Article Snippet: For western blotting, mouse anti-RACK1, rabbit anti-pY394Lck (Santa Cruz), anti-pY505Lck (Cell signaling), mouse anti-Lck (3A5), and phosphotyrosine-specific platinum 4G10 mAb (Millipore) were used.

    Techniques: Derivative Assay, Transgenic Assay, Mouse Assay, Microscopy, Fluorescence, Expressing

    The kinetics of Lck and RACK1 co-redistribution into forming immunological synapses (IS) in Jurkat T-cells . (A) Jurkat T-cells expressing RACK1–EGFP (green) were mixed with SEE-pulsed RAJI B-cells (denoted by the dotted circle), and the redistribution of RACK1 was observed by live cell imaging microscopy. Sequential time-lapse fluorescence microphotographs from one representative movie (Video S1 in Supplementary Material) are shown. (B) Statistical analysis of the kinetics of the relative recruitment index (RRI) of RACK1 to the forming IS measured after cell contact initiation ( n = 20). (C) The bar graph shows the time distribution of RACK1 residency in IS; the start and end points of RACK1 residency are marked by two white arrows shown in (A) ; n = 20 cells. (D) Lck-deficient JCAM1.6 Jurkat T-cells co-expressing Lck–CFP (red) and RACK1–mCitrine (green) constructs were mixed with SEE-pulsed RAJI B-cells (blue). Their redistribution during the formation of IS was observed by live cell imaging microscopy. Sequential time-lapse fluorescence microphotographs from one representative movie (Video S2 in Supplementary Material) are shown. An arrow points to the forming IS. (E) Statistical analysis of the kinetics of the relative recruitment index (RRI) of RACK1 and Lck to the forming IS measured after cell contact initiation ( n = 20).

    Journal: Frontiers in Immunology

    Article Title: TCR Triggering Induces the Formation of Lck–RACK1–Actinin-1 Multiprotein Network Affecting Lck Redistribution

    doi: 10.3389/fimmu.2016.00449

    Figure Lengend Snippet: The kinetics of Lck and RACK1 co-redistribution into forming immunological synapses (IS) in Jurkat T-cells . (A) Jurkat T-cells expressing RACK1–EGFP (green) were mixed with SEE-pulsed RAJI B-cells (denoted by the dotted circle), and the redistribution of RACK1 was observed by live cell imaging microscopy. Sequential time-lapse fluorescence microphotographs from one representative movie (Video S1 in Supplementary Material) are shown. (B) Statistical analysis of the kinetics of the relative recruitment index (RRI) of RACK1 to the forming IS measured after cell contact initiation ( n = 20). (C) The bar graph shows the time distribution of RACK1 residency in IS; the start and end points of RACK1 residency are marked by two white arrows shown in (A) ; n = 20 cells. (D) Lck-deficient JCAM1.6 Jurkat T-cells co-expressing Lck–CFP (red) and RACK1–mCitrine (green) constructs were mixed with SEE-pulsed RAJI B-cells (blue). Their redistribution during the formation of IS was observed by live cell imaging microscopy. Sequential time-lapse fluorescence microphotographs from one representative movie (Video S2 in Supplementary Material) are shown. An arrow points to the forming IS. (E) Statistical analysis of the kinetics of the relative recruitment index (RRI) of RACK1 and Lck to the forming IS measured after cell contact initiation ( n = 20).

    Article Snippet: For western blotting, mouse anti-RACK1, rabbit anti-pY394Lck (Santa Cruz), anti-pY505Lck (Cell signaling), mouse anti-Lck (3A5), and phosphotyrosine-specific platinum 4G10 mAb (Millipore) were used.

    Techniques: Expressing, Live Cell Imaging, Microscopy, Fluorescence, Construct

    Antibody-mediated engagement of TCR/CD4 receptors induces RACK1–Lck complex formation in primary CD4 + T-cells . (A) CD4 + T-cells precoated or non-precoated with biotinylated anti-TCR and anti-CD4 mAbs (TCRβ/CD4) were co-aggregated, or not (0 s), with streptavidin for the indicated time. RACK1 immunoprecipitates were blotted against Lck and RACK1. The bar graph at bottom shows the relative amount of co-immunoprecipitated Lck after normalization to RACK1. (B) The graph plot presents the relative fold increase of co-immunoprecipitated Lck (10 s after activation) from six independent experiments. The statistical analysis presented as mean ± SD was performed using the Student’s two-tailed t -test, ** p

    Journal: Frontiers in Immunology

    Article Title: TCR Triggering Induces the Formation of Lck–RACK1–Actinin-1 Multiprotein Network Affecting Lck Redistribution

    doi: 10.3389/fimmu.2016.00449

    Figure Lengend Snippet: Antibody-mediated engagement of TCR/CD4 receptors induces RACK1–Lck complex formation in primary CD4 + T-cells . (A) CD4 + T-cells precoated or non-precoated with biotinylated anti-TCR and anti-CD4 mAbs (TCRβ/CD4) were co-aggregated, or not (0 s), with streptavidin for the indicated time. RACK1 immunoprecipitates were blotted against Lck and RACK1. The bar graph at bottom shows the relative amount of co-immunoprecipitated Lck after normalization to RACK1. (B) The graph plot presents the relative fold increase of co-immunoprecipitated Lck (10 s after activation) from six independent experiments. The statistical analysis presented as mean ± SD was performed using the Student’s two-tailed t -test, ** p

    Article Snippet: For western blotting, mouse anti-RACK1, rabbit anti-pY394Lck (Santa Cruz), anti-pY505Lck (Cell signaling), mouse anti-Lck (3A5), and phosphotyrosine-specific platinum 4G10 mAb (Millipore) were used.

    Techniques: Immunoprecipitation, Activation Assay, Two Tailed Test

    Identification of additional components of RACK1–Lck complexes . (A) RACK1 immunoprecipitates from non-activated (0 s) and activated (10 s) CD4 + T-cell samples, and beads alone, were probed with anti-pY antibody (4G10). The arrows point to areas that show readily detectable phosphoproteins co-immunoprecipitating with RACK1. In the coomassie blue-stained gel, the areas depicted by arrows from all three samples were extracted (except for the 55-kDa area) and subjected to MALDI TOF/TOF MS analysis. A selected list of size-related and identified proteins with their potential pY sites are shown in (B) . The bead sample was negative for these proteins. (C) The kinetics of complex formation between RACK1 and indicated proteins before (0) and at the indicated time points after TCRβ/CD4 co-aggregation. RACK1 immunoprecipitates were probed with anti-α-actinin-1, anti-GADS, anti-LASP, anti-Lck, and anti-RACK1. (D) CD4 + T-cells were treated (PP2+) or not with PP2 inhibitor, activated, and lysed in TNE lysis buffer. RACK1 was immunoprecipitated, and samples were blotted against Lck, pY394 Lck , α-actinin-1, and RACK1. (E) Statistical analysis of (D) , actinin-1 panel, represents the relative fold change of RACK1 co-immunoprecipitated actinin-1 normalized to total RACK1. The statistical analysis presented as mean ± SD was performed using the Student’s two-tailed t -test, * p

    Journal: Frontiers in Immunology

    Article Title: TCR Triggering Induces the Formation of Lck–RACK1–Actinin-1 Multiprotein Network Affecting Lck Redistribution

    doi: 10.3389/fimmu.2016.00449

    Figure Lengend Snippet: Identification of additional components of RACK1–Lck complexes . (A) RACK1 immunoprecipitates from non-activated (0 s) and activated (10 s) CD4 + T-cell samples, and beads alone, were probed with anti-pY antibody (4G10). The arrows point to areas that show readily detectable phosphoproteins co-immunoprecipitating with RACK1. In the coomassie blue-stained gel, the areas depicted by arrows from all three samples were extracted (except for the 55-kDa area) and subjected to MALDI TOF/TOF MS analysis. A selected list of size-related and identified proteins with their potential pY sites are shown in (B) . The bead sample was negative for these proteins. (C) The kinetics of complex formation between RACK1 and indicated proteins before (0) and at the indicated time points after TCRβ/CD4 co-aggregation. RACK1 immunoprecipitates were probed with anti-α-actinin-1, anti-GADS, anti-LASP, anti-Lck, and anti-RACK1. (D) CD4 + T-cells were treated (PP2+) or not with PP2 inhibitor, activated, and lysed in TNE lysis buffer. RACK1 was immunoprecipitated, and samples were blotted against Lck, pY394 Lck , α-actinin-1, and RACK1. (E) Statistical analysis of (D) , actinin-1 panel, represents the relative fold change of RACK1 co-immunoprecipitated actinin-1 normalized to total RACK1. The statistical analysis presented as mean ± SD was performed using the Student’s two-tailed t -test, * p

    Article Snippet: For western blotting, mouse anti-RACK1, rabbit anti-pY394Lck (Santa Cruz), anti-pY505Lck (Cell signaling), mouse anti-Lck (3A5), and phosphotyrosine-specific platinum 4G10 mAb (Millipore) were used.

    Techniques: Staining, Mass Spectrometry, Lysis, Immunoprecipitation, Two Tailed Test

    Subcellular distribution of Lck and RACK1 in primary CD4 + T-cell . Fixed CD4 + T-cells were stained for RACK1 (green), Lck (red), and nuclei (blue) and visualized by confocal microscopy (A) or super-resolution N-SIM microscopy (B) . (C) Fluorescence intensity profile plot of Lck (red) and RACK1 (green) along the dotted line shown in the merged image of figure (B) . (D) Statistical analysis of the concentric juxtaposition of Lck and RACK1, which shows a larger distance of Lck from the cell centroid to its periphery ( n = 30), p ≤ 0.0001. (E) Magnification of the rectangle inset from the Merge image presented in (B) showing a subconcentrical juxtaposition of RACK1 to membrane-bound Lck. (F) The bar graph represents the statistical analysis of Lck and RACK1 colocalization using Pearson’s colocalization coefficient ( n = 20 cells). Error bars denote SD.

    Journal: Frontiers in Immunology

    Article Title: TCR Triggering Induces the Formation of Lck–RACK1–Actinin-1 Multiprotein Network Affecting Lck Redistribution

    doi: 10.3389/fimmu.2016.00449

    Figure Lengend Snippet: Subcellular distribution of Lck and RACK1 in primary CD4 + T-cell . Fixed CD4 + T-cells were stained for RACK1 (green), Lck (red), and nuclei (blue) and visualized by confocal microscopy (A) or super-resolution N-SIM microscopy (B) . (C) Fluorescence intensity profile plot of Lck (red) and RACK1 (green) along the dotted line shown in the merged image of figure (B) . (D) Statistical analysis of the concentric juxtaposition of Lck and RACK1, which shows a larger distance of Lck from the cell centroid to its periphery ( n = 30), p ≤ 0.0001. (E) Magnification of the rectangle inset from the Merge image presented in (B) showing a subconcentrical juxtaposition of RACK1 to membrane-bound Lck. (F) The bar graph represents the statistical analysis of Lck and RACK1 colocalization using Pearson’s colocalization coefficient ( n = 20 cells). Error bars denote SD.

    Article Snippet: For western blotting, mouse anti-RACK1, rabbit anti-pY394Lck (Santa Cruz), anti-pY505Lck (Cell signaling), mouse anti-Lck (3A5), and phosphotyrosine-specific platinum 4G10 mAb (Millipore) were used.

    Techniques: Staining, Confocal Microscopy, Microscopy, Fluorescence

    Model for the Rab10-EHBP1-EHD2 complex in mediating the autophagic engulfment of an LD during lipophagy. Following Rab7-mediated recruitment of degradative machinery to the LD surface, Rab10 works in a complex together with EHD2 and EHBP1 to promote extension of an LC3-positive autophagic membrane along the circumference of the LD surface.

    Journal: Science Advances

    Article Title: A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets

    doi: 10.1126/sciadv.1601470

    Figure Lengend Snippet: Model for the Rab10-EHBP1-EHD2 complex in mediating the autophagic engulfment of an LD during lipophagy. Following Rab7-mediated recruitment of degradative machinery to the LD surface, Rab10 works in a complex together with EHD2 and EHBP1 to promote extension of an LC3-positive autophagic membrane along the circumference of the LD surface.

    Article Snippet: After SDS-PAGE and immunoblotting, Rab10 was detected with an antibody against Rab10 (Sigma-Aldrich).

    Techniques:

    The Rab10-EHBP1-EHD2 complex mediates the engulfment of LDs by autophagic organelles. ( A ) Live-cell confocal fluorescence microscopy of two distinct LDs (stained with MDH, blue) from Hep3B cells expressing either mCherry-Rab10 (top series) or GFP-Rab10 (lower series). Imaging reveals the association of LD-bound Rab10 at early time points with phagophore/autophagosome-associated Rab10 (0 s) extending to nearly surround the perimeter of LDs at later time points. Dashed outlines provide fiducial points of reference as the envelopment of the LD by the phagophore progresses. These events are representative of data from more than 30 individual cells examined by live-cell imaging. ( B and C ) Quantification of the percentage of LDs associated with LC3- or Atg16L1-positive structures in Hep3B hepatoma cells after 48-hour siRNA treatment with the indicated siRNAs (Tri-siRNA, triple knockdown). Cells were preloaded with 150 μM oleic acid overnight. ( D and E ) Quantification of the percentage of LDs associated with LC3- or Atg16L1-positive structures after culture in low-serum conditions in WT or Rab10 KO MEFs. Cells were preloaded with 400 μM oleic acid overnight. ( F ) Quantification of the percentage of LDs associated with LAMP1-positive structures after 48-hour siRNA treatment followed by 48-hour starvation from n = 3 independent experiments, measuring 20 cells per condition. Cells were preloaded with 150 μM oleic acid overnight. ( G and H ) LDs visualized from WT or Rab10 KO MEFs were divided into three groups on the basis of their association with LAMP-1: “none,” “attached,” or “engulfed” (G). Manual counting of LDs (H) in each group from WT and Rab10 KO MEFs. The graphs represent observations from n = 3 independent experiments, measuring 20 cells per condition. Cells were preloaded with 400 μM oleic acid overnight. Data are represented as means ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Scale bars, 1 μm.

    Journal: Science Advances

    Article Title: A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets

    doi: 10.1126/sciadv.1601470

    Figure Lengend Snippet: The Rab10-EHBP1-EHD2 complex mediates the engulfment of LDs by autophagic organelles. ( A ) Live-cell confocal fluorescence microscopy of two distinct LDs (stained with MDH, blue) from Hep3B cells expressing either mCherry-Rab10 (top series) or GFP-Rab10 (lower series). Imaging reveals the association of LD-bound Rab10 at early time points with phagophore/autophagosome-associated Rab10 (0 s) extending to nearly surround the perimeter of LDs at later time points. Dashed outlines provide fiducial points of reference as the envelopment of the LD by the phagophore progresses. These events are representative of data from more than 30 individual cells examined by live-cell imaging. ( B and C ) Quantification of the percentage of LDs associated with LC3- or Atg16L1-positive structures in Hep3B hepatoma cells after 48-hour siRNA treatment with the indicated siRNAs (Tri-siRNA, triple knockdown). Cells were preloaded with 150 μM oleic acid overnight. ( D and E ) Quantification of the percentage of LDs associated with LC3- or Atg16L1-positive structures after culture in low-serum conditions in WT or Rab10 KO MEFs. Cells were preloaded with 400 μM oleic acid overnight. ( F ) Quantification of the percentage of LDs associated with LAMP1-positive structures after 48-hour siRNA treatment followed by 48-hour starvation from n = 3 independent experiments, measuring 20 cells per condition. Cells were preloaded with 150 μM oleic acid overnight. ( G and H ) LDs visualized from WT or Rab10 KO MEFs were divided into three groups on the basis of their association with LAMP-1: “none,” “attached,” or “engulfed” (G). Manual counting of LDs (H) in each group from WT and Rab10 KO MEFs. The graphs represent observations from n = 3 independent experiments, measuring 20 cells per condition. Cells were preloaded with 400 μM oleic acid overnight. Data are represented as means ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Scale bars, 1 μm.

    Article Snippet: After SDS-PAGE and immunoblotting, Rab10 was detected with an antibody against Rab10 (Sigma-Aldrich).

    Techniques: Fluorescence, Microscopy, Staining, Expressing, Imaging, Live Cell Imaging, Gene Knockout

    Rab10 acts downstream of Rab7 to facilitate the autophagic degradation of LDs. ( A and B ) Immunoblot of GST-EHBP1– or GST-RILP–mediated pulldowns for Rab7 (A) or Rab10 (B) in Hep3B hepatoma cells. Rab7 exhibits a specific affinity for RILP, whereas Rab10 interacts with both EHBP1 and RILP. ( C ) Representative immunoblotting of the results for GST or GST-RILP pulldowns of Rab7 in WT or Rab10 KO MEFs, showing that Rab10 KO does not affect the binding of Rab7 to RILP. ( D ) Immunoblotting of subcellular density gradient fractions of Hep3B cells following serum starvation in HBSS for 2 hours, followed by flotation of a CLF through an 8 to 27% discontinuous OptiPrep gradient. Boxes indicate distinct peak density fractions for either Rab10 (blue box, fraction 2) or Rab7 (red box, fraction 4). ( E ) Live-cell confocal fluorescence microscopy of a HuH-7 hepatoma cell cotransfected with both GFP-Rab7 and mCherry-Rab10 and subjected to serum starvation. LDs are stained with MDH (blue). Arrows indicate the extension of a Rab7-positive membrane away from the LD and the subsequent recruitment of a Rab10-positive structure (arrowheads) to the LD. ( F ) Quantification of the average percentage of LDs positive for the presence of Rab10 alone, Rab7 alone, or both Rab10 and Rab7 from n = 10 cells. ( G ) Quantification of the average number of Rab10-positive LDs in resting or serum-starved HuH-7 hepatoma cells treated with NT siRNA or Rab7 siRNA. ( H ) Quantification of the average number of Rab7-positive LDs in resting or serum-starved HuH-7 cells treated with NT siRNA or Rab10 siRNA. Data represent the means from n = 3 independent experiments, measuring > 50 cells per condition per repeat. *** P ≤ 0.001. Scale bars, 1 μm.

    Journal: Science Advances

    Article Title: A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets

    doi: 10.1126/sciadv.1601470

    Figure Lengend Snippet: Rab10 acts downstream of Rab7 to facilitate the autophagic degradation of LDs. ( A and B ) Immunoblot of GST-EHBP1– or GST-RILP–mediated pulldowns for Rab7 (A) or Rab10 (B) in Hep3B hepatoma cells. Rab7 exhibits a specific affinity for RILP, whereas Rab10 interacts with both EHBP1 and RILP. ( C ) Representative immunoblotting of the results for GST or GST-RILP pulldowns of Rab7 in WT or Rab10 KO MEFs, showing that Rab10 KO does not affect the binding of Rab7 to RILP. ( D ) Immunoblotting of subcellular density gradient fractions of Hep3B cells following serum starvation in HBSS for 2 hours, followed by flotation of a CLF through an 8 to 27% discontinuous OptiPrep gradient. Boxes indicate distinct peak density fractions for either Rab10 (blue box, fraction 2) or Rab7 (red box, fraction 4). ( E ) Live-cell confocal fluorescence microscopy of a HuH-7 hepatoma cell cotransfected with both GFP-Rab7 and mCherry-Rab10 and subjected to serum starvation. LDs are stained with MDH (blue). Arrows indicate the extension of a Rab7-positive membrane away from the LD and the subsequent recruitment of a Rab10-positive structure (arrowheads) to the LD. ( F ) Quantification of the average percentage of LDs positive for the presence of Rab10 alone, Rab7 alone, or both Rab10 and Rab7 from n = 10 cells. ( G ) Quantification of the average number of Rab10-positive LDs in resting or serum-starved HuH-7 hepatoma cells treated with NT siRNA or Rab7 siRNA. ( H ) Quantification of the average number of Rab7-positive LDs in resting or serum-starved HuH-7 cells treated with NT siRNA or Rab10 siRNA. Data represent the means from n = 3 independent experiments, measuring > 50 cells per condition per repeat. *** P ≤ 0.001. Scale bars, 1 μm.

    Article Snippet: After SDS-PAGE and immunoblotting, Rab10 was detected with an antibody against Rab10 (Sigma-Aldrich).

    Techniques: Gene Knockout, Binding Assay, Fluorescence, Microscopy, Staining

    Serum starvation potentiates the formation of a Rab10 complex together with the membrane trafficking proteins EHBP1 and EHD2 at the lipophagic junction. ( A ) Immunoblot analysis of Hep3B cells transfected to express HA-EHBP1, lysed, and subjected to a GST-Rab10 pulldown. ( B ) Immunoblot analysis of a GST pulldown experiment in Hep3B cells coexpressing a GST-tagged form of the Rab10-interacting domain of EHBP1 (residues 600 to 902) and HA-tagged Rab10-WT, Rab10-T23N, or Rab10-Q68L. ( C and D ) Representative immunoblots from pulldown experiments using the same GST-EHBP1 fragment to examine the effect of EHBP1 interactions with endogenous Rab10 after serum starvation (C) or treatment with Torin1 (D) ( n = 3 independent experiments for each condition). Numbers below the pulldown blot represent the mean fold enrichment in protein levels. ( E and F ) Representative immunoblots from GST-Rab10 pulldown experiments in Hep3B cells, probing for interactions between Rab10 and EHD2 under serum-starved (E) or Torin1-treated conditions (F) ( n = 3 independent experiments for each condition). Numbers below the pulldown blot represent the mean fold enrichment in protein levels. ( G ) Representative immunoblot of a GST-Rab10 pulldown of EHD2 in Hep3B cells previously treated with either siNT or siEHBP1 and subjected to HBSS starvation for 1 hour ( n = 3 independent experiments), quantified in ( H ). ( I ) Fluorescence images of EHBP1-positive LDs in HuH-7 cells expressing HA-EHBP1 and starved in HBSS for 1 hour before fixation and immunostaining with anti-HA antibody (green). Cells were preloaded with 150 μM oleic acid overnight. LDs are stained with ORO (red). ( J ) Fluorescence image of a HuH-7 cell transfected to express GFP-EHD2 (green) and mCherry-Rab10 (red) before serum starvation in HBSS for 1 hour and fixation. The boxed region shows a higher-magnification image of an example of an MDH-stained LD (blue) that is also positive for GFP-EHD2 and mCherry-Rab10. Scale bars, 10 μm. ( K and L ) Quantification of the appearance of GFP-Rab10–positive LDs (K) or GFP-EHD2–positive LDs (L) in HuH-7 cells following siRNA-mediated knockdown of EHBP1 and EHD2 (K) or EHBP1 and Rab10 (L) for 48 hours, followed by serum starvation in HBSS for 1 hour. Results are from n = 3 independent experiments each and are represented as mean ± SD. ** P ≤ 0.01; *** P ≤ 0.001. A total of 80 cells were quantified per condition. ( M ) Live-cell confocal fluorescence imaging of a starved Hep3B hepatoma cell coexpressing GFP-EHD2 and mCherry-Rab10, depicting the sequential recruitment of Rab10 and EHD2 to MDH-labeled LDs (blue). Note the presence of the mCherry-Rab10–positive structure at the periphery of the LD (arrowhead) before the recruitment of GFP-EHD2, resulting in the emergence of signal overlap by 35 min. Scale bars, 5 μm.

    Journal: Science Advances

    Article Title: A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets

    doi: 10.1126/sciadv.1601470

    Figure Lengend Snippet: Serum starvation potentiates the formation of a Rab10 complex together with the membrane trafficking proteins EHBP1 and EHD2 at the lipophagic junction. ( A ) Immunoblot analysis of Hep3B cells transfected to express HA-EHBP1, lysed, and subjected to a GST-Rab10 pulldown. ( B ) Immunoblot analysis of a GST pulldown experiment in Hep3B cells coexpressing a GST-tagged form of the Rab10-interacting domain of EHBP1 (residues 600 to 902) and HA-tagged Rab10-WT, Rab10-T23N, or Rab10-Q68L. ( C and D ) Representative immunoblots from pulldown experiments using the same GST-EHBP1 fragment to examine the effect of EHBP1 interactions with endogenous Rab10 after serum starvation (C) or treatment with Torin1 (D) ( n = 3 independent experiments for each condition). Numbers below the pulldown blot represent the mean fold enrichment in protein levels. ( E and F ) Representative immunoblots from GST-Rab10 pulldown experiments in Hep3B cells, probing for interactions between Rab10 and EHD2 under serum-starved (E) or Torin1-treated conditions (F) ( n = 3 independent experiments for each condition). Numbers below the pulldown blot represent the mean fold enrichment in protein levels. ( G ) Representative immunoblot of a GST-Rab10 pulldown of EHD2 in Hep3B cells previously treated with either siNT or siEHBP1 and subjected to HBSS starvation for 1 hour ( n = 3 independent experiments), quantified in ( H ). ( I ) Fluorescence images of EHBP1-positive LDs in HuH-7 cells expressing HA-EHBP1 and starved in HBSS for 1 hour before fixation and immunostaining with anti-HA antibody (green). Cells were preloaded with 150 μM oleic acid overnight. LDs are stained with ORO (red). ( J ) Fluorescence image of a HuH-7 cell transfected to express GFP-EHD2 (green) and mCherry-Rab10 (red) before serum starvation in HBSS for 1 hour and fixation. The boxed region shows a higher-magnification image of an example of an MDH-stained LD (blue) that is also positive for GFP-EHD2 and mCherry-Rab10. Scale bars, 10 μm. ( K and L ) Quantification of the appearance of GFP-Rab10–positive LDs (K) or GFP-EHD2–positive LDs (L) in HuH-7 cells following siRNA-mediated knockdown of EHBP1 and EHD2 (K) or EHBP1 and Rab10 (L) for 48 hours, followed by serum starvation in HBSS for 1 hour. Results are from n = 3 independent experiments each and are represented as mean ± SD. ** P ≤ 0.01; *** P ≤ 0.001. A total of 80 cells were quantified per condition. ( M ) Live-cell confocal fluorescence imaging of a starved Hep3B hepatoma cell coexpressing GFP-EHD2 and mCherry-Rab10, depicting the sequential recruitment of Rab10 and EHD2 to MDH-labeled LDs (blue). Note the presence of the mCherry-Rab10–positive structure at the periphery of the LD (arrowhead) before the recruitment of GFP-EHD2, resulting in the emergence of signal overlap by 35 min. Scale bars, 5 μm.

    Article Snippet: After SDS-PAGE and immunoblotting, Rab10 was detected with an antibody against Rab10 (Sigma-Aldrich).

    Techniques: Transfection, Hemagglutination Assay, Western Blot, Fluorescence, Expressing, Immunostaining, Staining, Imaging, Labeling

    Rab10 distributes to LD-associated structures upon serum starvation. ( A ) Western blot analysis of a purified LD preparation isolated from HuH-7 hepatoma cells, probed with antibodies targeting a variety of organelle markers, including LAMP1 (lysosome), protein disulfide isomerase (PDI) (ER), PLIN2 (LD), and Rab10. Lanes indicate the whole-cell lysate (WCL), postnuclear supernatant (PNS), and LD fraction (LD). ( B ) Expression of sfGFP-tagged Rab10 or ( C ) antibody staining of endogenous Rab10 in oleate-loaded Hep3B cells reveals the presence of prominent Rab10-positive structures (green) in close proximity to ORO-stained LDs (red). ( D and E ) Fluorescence microscopy images of serum-starved HuH-7 hepatoma cells expressing either the constitutively active sfGFP-Q68L (D) or dominant-negative sfGFP-T23N (E) mutant forms of Rab10. Arrowheads indicate examples of Rab10-positive LD-associated structures. ( F ) Quantification of the number of Rab10-positive LDs observed in n = 3 independent experiments performed on control or HBSS-starved cells (100 cells per condition). *** P ≤ 0.001. ( G to I ) Comparison of images of a solitary sfGFP-Rab10–positive LD (from a HuH-7 cell HBSS-starved for 1 hour) observed using conventional wide-field epifluorescence (G) or super-resolution microscopy (H and I). The higher resolution of the latter two images (H and I) reveals a membranous structure (I) (sfGFP signal alone) that appears to extend completely around the LD surface. Scale bars, 10 μm (B and C), 5 μm (D and E), and 1 μm (G to I).

    Journal: Science Advances

    Article Title: A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets

    doi: 10.1126/sciadv.1601470

    Figure Lengend Snippet: Rab10 distributes to LD-associated structures upon serum starvation. ( A ) Western blot analysis of a purified LD preparation isolated from HuH-7 hepatoma cells, probed with antibodies targeting a variety of organelle markers, including LAMP1 (lysosome), protein disulfide isomerase (PDI) (ER), PLIN2 (LD), and Rab10. Lanes indicate the whole-cell lysate (WCL), postnuclear supernatant (PNS), and LD fraction (LD). ( B ) Expression of sfGFP-tagged Rab10 or ( C ) antibody staining of endogenous Rab10 in oleate-loaded Hep3B cells reveals the presence of prominent Rab10-positive structures (green) in close proximity to ORO-stained LDs (red). ( D and E ) Fluorescence microscopy images of serum-starved HuH-7 hepatoma cells expressing either the constitutively active sfGFP-Q68L (D) or dominant-negative sfGFP-T23N (E) mutant forms of Rab10. Arrowheads indicate examples of Rab10-positive LD-associated structures. ( F ) Quantification of the number of Rab10-positive LDs observed in n = 3 independent experiments performed on control or HBSS-starved cells (100 cells per condition). *** P ≤ 0.001. ( G to I ) Comparison of images of a solitary sfGFP-Rab10–positive LD (from a HuH-7 cell HBSS-starved for 1 hour) observed using conventional wide-field epifluorescence (G) or super-resolution microscopy (H and I). The higher resolution of the latter two images (H and I) reveals a membranous structure (I) (sfGFP signal alone) that appears to extend completely around the LD surface. Scale bars, 10 μm (B and C), 5 μm (D and E), and 1 μm (G to I).

    Article Snippet: After SDS-PAGE and immunoblotting, Rab10 was detected with an antibody against Rab10 (Sigma-Aldrich).

    Techniques: Western Blot, Purification, Isolation, Expressing, Staining, Fluorescence, Microscopy, Dominant Negative Mutation, Mutagenesis

    Rab10 function is required for LD catabolism. ( A ) Representative immunoblot from n = 3 independent experiments showing the efficiency ( > 90%) of Rab10 siRNA knockdown in HuH-7 hepatoma cells. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. ( B and C ) Fluorescence imaging of HuH-7 cells subjected to 48-hour treatment with either control nontargeting (NT) siRNA or Rab10-directed siRNA and subsequently starved for an additional 48 hours. LDs are stained with ORO, and nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI). Total ORO-stained area per cell was quantified in (C) from n = 3 independent experiments ( > 350 cells measured per experiment). ( D ) HuH-7 cells were treated with control NT siRNA or Rab10 siRNA for 48 hours before re-expression of GFP-tagged forms of wild-type (WT), active (−Q68L), or inactive (−T23N) forms of Rab10. Cells were then serum-starved for a period of 48 hours to look for rescue of the LD breakdown phenotype (total ORO-stained area quantified in n = 3 independent experiments, 30 cells per repeat). ( E ) Quantification of a similar knockdown/re-expression experiment performed in (D), with the exception that the lysosomal protease inhibitor CQ was included in the medium during the starvation period ( n = 3 independent experiments, 25 cells measured per repeat). ( F to I ) LDs accumulate in cells isolated from Rab10 KO mice. Cells were preloaded with 400 μM oleic acid overnight. (F) Representative immunoblot of MEFs isolated from WT or Rab10 KO embryos. (G) Measurement of total triglyceride content in WT or Rab10 KO MEFs. (H) Representative fluorescence images of oleate-loaded WT or Rab10 KO MEFs, stained with ORO and DAPI. (I) Digital quantification of average LD content per cell in WT or Rab10 KO MEFs ( n = 3 independent experiments, 300 cells per repeat). ( J ) Quantification of the total LD area per cell in Rab10 KO MEFs transfected with either GFP vector alone or GFP-Rab10 ( n = 3 independent experiments, 400 cells per repeat). Cells were preloaded with 400 μM oleic acid overnight. ( K ) Quantification of the release of [ 3 H]H 2 O into the medium as a functional readout of mitochondrial β-oxidation in either WT or Rab10 MEFs subjected to 6 hours of growth under full-serum or serum-starved conditions. Cells were pulse-labeled with [9,10- 3 H]oleic acid. Data are represented as means ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; N.S., not significant. Scale bars, 10 μm.

    Journal: Science Advances

    Article Title: A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets

    doi: 10.1126/sciadv.1601470

    Figure Lengend Snippet: Rab10 function is required for LD catabolism. ( A ) Representative immunoblot from n = 3 independent experiments showing the efficiency ( > 90%) of Rab10 siRNA knockdown in HuH-7 hepatoma cells. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. ( B and C ) Fluorescence imaging of HuH-7 cells subjected to 48-hour treatment with either control nontargeting (NT) siRNA or Rab10-directed siRNA and subsequently starved for an additional 48 hours. LDs are stained with ORO, and nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI). Total ORO-stained area per cell was quantified in (C) from n = 3 independent experiments ( > 350 cells measured per experiment). ( D ) HuH-7 cells were treated with control NT siRNA or Rab10 siRNA for 48 hours before re-expression of GFP-tagged forms of wild-type (WT), active (−Q68L), or inactive (−T23N) forms of Rab10. Cells were then serum-starved for a period of 48 hours to look for rescue of the LD breakdown phenotype (total ORO-stained area quantified in n = 3 independent experiments, 30 cells per repeat). ( E ) Quantification of a similar knockdown/re-expression experiment performed in (D), with the exception that the lysosomal protease inhibitor CQ was included in the medium during the starvation period ( n = 3 independent experiments, 25 cells measured per repeat). ( F to I ) LDs accumulate in cells isolated from Rab10 KO mice. Cells were preloaded with 400 μM oleic acid overnight. (F) Representative immunoblot of MEFs isolated from WT or Rab10 KO embryos. (G) Measurement of total triglyceride content in WT or Rab10 KO MEFs. (H) Representative fluorescence images of oleate-loaded WT or Rab10 KO MEFs, stained with ORO and DAPI. (I) Digital quantification of average LD content per cell in WT or Rab10 KO MEFs ( n = 3 independent experiments, 300 cells per repeat). ( J ) Quantification of the total LD area per cell in Rab10 KO MEFs transfected with either GFP vector alone or GFP-Rab10 ( n = 3 independent experiments, 400 cells per repeat). Cells were preloaded with 400 μM oleic acid overnight. ( K ) Quantification of the release of [ 3 H]H 2 O into the medium as a functional readout of mitochondrial β-oxidation in either WT or Rab10 MEFs subjected to 6 hours of growth under full-serum or serum-starved conditions. Cells were pulse-labeled with [9,10- 3 H]oleic acid. Data are represented as means ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; N.S., not significant. Scale bars, 10 μm.

    Article Snippet: After SDS-PAGE and immunoblotting, Rab10 was detected with an antibody against Rab10 (Sigma-Aldrich).

    Techniques: Fluorescence, Imaging, Staining, Expressing, Protease Inhibitor, Isolation, Gene Knockout, Mouse Assay, Transfection, Plasmid Preparation, Functional Assay, Labeling

    Rab10 is recruited to the LD during starvation-induced autophagy. ( A to D ) Fluorescence images of HuH-7 hepatoma cells expressing sfGFP-Rab10 under resting (A), HBSS-starved (B), Torin1-treated (C), or 3-MA–treated (D) conditions. LDs are stained with ORO (red). Rab10-positive LDs are indicated by arrowheads, and average numbers of Rab10-positive LDs per cell are quantified in ( E ) ( n = 3 independent experiments, 100 cells counted per condition). ( F ) Quantification of the effect of 3-MA treatment on resting or serum-starved HuH-7 cells ( n = 3 independent cells, 100 cells counted per condition). ( G ) Results of an anti-HA immunoblot from a GTP-agarose bead pulldown of HA-tagged Rab10Q68L or HA-tagged Rab10T23N. ( H and I ) Immunoblots of Rab10 showing a differential association with GTP beads in HuH-7 cells subjected under resting versus HBSS starvation conditions for 1 hour (H) or treated with DMSO (dimethyl sulfoxide) or 1 μM Torin1 for 1 hour (I). ( J ) Quantification of Rab10 protein levels from (H) and (I) ( n = 3 independent experiments). Data are represented as means ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Scale bars, 1 μm.

    Journal: Science Advances

    Article Title: A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets

    doi: 10.1126/sciadv.1601470

    Figure Lengend Snippet: Rab10 is recruited to the LD during starvation-induced autophagy. ( A to D ) Fluorescence images of HuH-7 hepatoma cells expressing sfGFP-Rab10 under resting (A), HBSS-starved (B), Torin1-treated (C), or 3-MA–treated (D) conditions. LDs are stained with ORO (red). Rab10-positive LDs are indicated by arrowheads, and average numbers of Rab10-positive LDs per cell are quantified in ( E ) ( n = 3 independent experiments, 100 cells counted per condition). ( F ) Quantification of the effect of 3-MA treatment on resting or serum-starved HuH-7 cells ( n = 3 independent cells, 100 cells counted per condition). ( G ) Results of an anti-HA immunoblot from a GTP-agarose bead pulldown of HA-tagged Rab10Q68L or HA-tagged Rab10T23N. ( H and I ) Immunoblots of Rab10 showing a differential association with GTP beads in HuH-7 cells subjected under resting versus HBSS starvation conditions for 1 hour (H) or treated with DMSO (dimethyl sulfoxide) or 1 μM Torin1 for 1 hour (I). ( J ) Quantification of Rab10 protein levels from (H) and (I) ( n = 3 independent experiments). Data are represented as means ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Scale bars, 1 μm.

    Article Snippet: After SDS-PAGE and immunoblotting, Rab10 was detected with an antibody against Rab10 (Sigma-Aldrich).

    Techniques: Fluorescence, Expressing, Staining, Hemagglutination Assay, Western Blot

    Rab10-positive LD-associated structures represent nascent autophagic organelles. ( A to E ) Fluorescence images of Hep3B hepatoma cells comparing the colocalization of autophagic and organelle markers with LD-localized Rab10. Boxed areas represent regions of higher magnification. Cells were transfected for 24 hours with mCherry-Rab10 or sfGFP-Rab10, preloaded with 150 μM oleic acid overnight, serum-starved in HBSS for 1 hour, and stained with the LD dye monodansylpentane (MDH) and an antibody to LC3 (A) or transfected to express the autophagic marker GFP-Atg16L1 (B), Rab11 (C), the ER marker Sec61β (D), or the lysosomal marker LAMP1-mCherry (E). Inset number represents the frequency of LD-associated marker that colocalizes with Rab10 in HBSS-starved cells averaged from three independent experiments. Scale bars, 10 μm. ( F ) Subcellular density gradient fractionation of oleate-loaded Hep3B cells starved in HBSS for 2 hours, lysed (WCL), and further separated into a PNS, CLF, and HSS (high-speed supernatant). The CLF was subsequently loaded onto an 8 to 27% discontinuous iodixanol (OptiPrep) gradient for separation by ultracentrifugation. Nine fractions were collected from the top of the gradient and blotted for Rab10, a mitochondrial marker (αCOXIV), an endoplasmic reticulum marker (PDI), and the lysosomal resident protein LAMP1.

    Journal: Science Advances

    Article Title: A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets

    doi: 10.1126/sciadv.1601470

    Figure Lengend Snippet: Rab10-positive LD-associated structures represent nascent autophagic organelles. ( A to E ) Fluorescence images of Hep3B hepatoma cells comparing the colocalization of autophagic and organelle markers with LD-localized Rab10. Boxed areas represent regions of higher magnification. Cells were transfected for 24 hours with mCherry-Rab10 or sfGFP-Rab10, preloaded with 150 μM oleic acid overnight, serum-starved in HBSS for 1 hour, and stained with the LD dye monodansylpentane (MDH) and an antibody to LC3 (A) or transfected to express the autophagic marker GFP-Atg16L1 (B), Rab11 (C), the ER marker Sec61β (D), or the lysosomal marker LAMP1-mCherry (E). Inset number represents the frequency of LD-associated marker that colocalizes with Rab10 in HBSS-starved cells averaged from three independent experiments. Scale bars, 10 μm. ( F ) Subcellular density gradient fractionation of oleate-loaded Hep3B cells starved in HBSS for 2 hours, lysed (WCL), and further separated into a PNS, CLF, and HSS (high-speed supernatant). The CLF was subsequently loaded onto an 8 to 27% discontinuous iodixanol (OptiPrep) gradient for separation by ultracentrifugation. Nine fractions were collected from the top of the gradient and blotted for Rab10, a mitochondrial marker (αCOXIV), an endoplasmic reticulum marker (PDI), and the lysosomal resident protein LAMP1.

    Article Snippet: After SDS-PAGE and immunoblotting, Rab10 was detected with an antibody against Rab10 (Sigma-Aldrich).

    Techniques: Fluorescence, Transfection, Staining, Marker, Fractionation

    EHD2 and EHBP1 are involved in Rab10-mediated LD breakdown. ( A and B ) Representative immunoblots showing the efficiency of a 48-hour EHD2 (A) or EHBP1 (B) siRNA–mediated knockdown in the Hep3B hepatoma cell line. ( C ) Quantification of the effect of EHD2 or EHBP1 knockdown on LD breakdown in Hep3B cells following 48-hour knockdown and 48-hour serum starvation. Average ORO-stained LD area (in pixels) per cell was calculated from n = 3 independent experiments in 100 cells per condition. Cells were preloaded with 150 μM oleic acid overnight. ( D ) The dependence of LD catabolism on EHD2 activity was examined by depleting Hep3B cells of EHD2 by siRNA treatment for 24 hours and then transfecting them with GFP alone, GPF-EHD2, or GFP-EHD2-T72A (ATPase-dead) for an additional 24 hours in the presence or absence of CQ. LD breakdown is represented as the average ORO-stained area per cell from n = 3 experiments in 25 transfected cells per condition. Cells were preloaded with 150 μM oleic acid overnight. ( E ) Subcellular fractionation of oleate-loaded Hep3B cells starved for 2 hours in HBSS through a 0 to 30% discontinuous iodixanol (OptiPrep) gradient. Fractions were collected from the top of the gradient and blotted for EHD2, the endosomal marker Rab5, or the lysosomal marker LAMP1. ( F ) Representative fluorescence images of basal or serum-starved Hep3B cells treated with siNT, Rab10 siRNA, or EHD2 siRNA and expressing a dual-fluorescent red fluorescent protien (RFP)–GFP–PLIN2 construct that had been serum-starved for 24 hours to measure the appearance of “RFP-only” PLIN2-positive puncta, indicative of interactions between the LD and the acidic lysosomal compartment. Cells were preloaded with 150 μM oleic acid overnight. ( G ) Quantification of the number of “RFP-only” PLIN2-positive puncta per Hep3B cell, reflective of active lipophagy, from n = 3 independent experiments, measuring 22 transfected cells per condition. The data are represented as mean ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Scale bars, 10 μm.

    Journal: Science Advances

    Article Title: A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets

    doi: 10.1126/sciadv.1601470

    Figure Lengend Snippet: EHD2 and EHBP1 are involved in Rab10-mediated LD breakdown. ( A and B ) Representative immunoblots showing the efficiency of a 48-hour EHD2 (A) or EHBP1 (B) siRNA–mediated knockdown in the Hep3B hepatoma cell line. ( C ) Quantification of the effect of EHD2 or EHBP1 knockdown on LD breakdown in Hep3B cells following 48-hour knockdown and 48-hour serum starvation. Average ORO-stained LD area (in pixels) per cell was calculated from n = 3 independent experiments in 100 cells per condition. Cells were preloaded with 150 μM oleic acid overnight. ( D ) The dependence of LD catabolism on EHD2 activity was examined by depleting Hep3B cells of EHD2 by siRNA treatment for 24 hours and then transfecting them with GFP alone, GPF-EHD2, or GFP-EHD2-T72A (ATPase-dead) for an additional 24 hours in the presence or absence of CQ. LD breakdown is represented as the average ORO-stained area per cell from n = 3 experiments in 25 transfected cells per condition. Cells were preloaded with 150 μM oleic acid overnight. ( E ) Subcellular fractionation of oleate-loaded Hep3B cells starved for 2 hours in HBSS through a 0 to 30% discontinuous iodixanol (OptiPrep) gradient. Fractions were collected from the top of the gradient and blotted for EHD2, the endosomal marker Rab5, or the lysosomal marker LAMP1. ( F ) Representative fluorescence images of basal or serum-starved Hep3B cells treated with siNT, Rab10 siRNA, or EHD2 siRNA and expressing a dual-fluorescent red fluorescent protien (RFP)–GFP–PLIN2 construct that had been serum-starved for 24 hours to measure the appearance of “RFP-only” PLIN2-positive puncta, indicative of interactions between the LD and the acidic lysosomal compartment. Cells were preloaded with 150 μM oleic acid overnight. ( G ) Quantification of the number of “RFP-only” PLIN2-positive puncta per Hep3B cell, reflective of active lipophagy, from n = 3 independent experiments, measuring 22 transfected cells per condition. The data are represented as mean ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Scale bars, 10 μm.

    Article Snippet: After SDS-PAGE and immunoblotting, Rab10 was detected with an antibody against Rab10 (Sigma-Aldrich).

    Techniques: Western Blot, Staining, Activity Assay, Transfection, Fractionation, Marker, Fluorescence, Expressing, Construct

    EM reveals that Rab10-associated autophagosomes extend membrane to envelop adjacent LDs. ( A and B ) Low-magnification EM of LD-autophagosome interactions in HuH-7 cells that were transfected to express an active Q68L form of GFP-tagged Rab10 before starvation in HBSS for 1 hour, followed by fixation and embedding. Scale bars, 1 μm. ( A′ and B′ ) Higher magnification of boxed regions shows autophagic membrane extensions (arrows) moving outward along the LDs during the envelopment process. ( C and D ) Corresponding low-magnification fluorescence (C) and electron (D) micrographs of a cell exhibiting several prominent Rab10-positive LDs reveal numerous LDs with and without associated Rab10-positive autophagic structures (scale bars, 2 μm). ( 1 to 3 ) Correlative EM images of HuH-7 cells expressing active GFP-Rab10 that were cultured on finder grids and photographed by phase and fluorescence microscopy to first locate and identify Rab10-LD–positive structures before fixation and embedding. Boxes show corresponding higher-magnification EM of these structures, confirming the intimate association of Rab10-positive membranes with the engulfed LDs. APM, autophagic membrane.

    Journal: Science Advances

    Article Title: A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets

    doi: 10.1126/sciadv.1601470

    Figure Lengend Snippet: EM reveals that Rab10-associated autophagosomes extend membrane to envelop adjacent LDs. ( A and B ) Low-magnification EM of LD-autophagosome interactions in HuH-7 cells that were transfected to express an active Q68L form of GFP-tagged Rab10 before starvation in HBSS for 1 hour, followed by fixation and embedding. Scale bars, 1 μm. ( A′ and B′ ) Higher magnification of boxed regions shows autophagic membrane extensions (arrows) moving outward along the LDs during the envelopment process. ( C and D ) Corresponding low-magnification fluorescence (C) and electron (D) micrographs of a cell exhibiting several prominent Rab10-positive LDs reveal numerous LDs with and without associated Rab10-positive autophagic structures (scale bars, 2 μm). ( 1 to 3 ) Correlative EM images of HuH-7 cells expressing active GFP-Rab10 that were cultured on finder grids and photographed by phase and fluorescence microscopy to first locate and identify Rab10-LD–positive structures before fixation and embedding. Boxes show corresponding higher-magnification EM of these structures, confirming the intimate association of Rab10-positive membranes with the engulfed LDs. APM, autophagic membrane.

    Article Snippet: After SDS-PAGE and immunoblotting, Rab10 was detected with an antibody against Rab10 (Sigma-Aldrich).

    Techniques: Electron Microscopy, Transfection, Fluorescence, Expressing, Cell Culture, Microscopy

    Kalirin-7 interacts with synphilin-1 in vitro and in vivo . (A) Mapping of the interacting domain in the kalirin-7 protein. FLAG-kalirin-7 constructs as shown in the diagram were co-transfected with V5-synphilin-1 in HEK293 cells. 24 h after transfection, cells were subjected to immunoprecipitation with anti-FLAG agarose beads and subsequently kalirin-7 and synphilin-1 immunoreactivities were monitored applying anti-FLAG- or anti-V5 antibodies, respectively. IP indicates antibodies used for pulling down target proteins. IB indicates antibodies used for detection in western blot. The figure shows that kalirin-7 co-immunopreciptates with synphilin-1 and that spectrin repeats III and IV of the kalirin-7 protein are crucial for the interaction. Quantification of kalirin-7 fragment expression is shown in Figure S3 . (B) Mapping of the binding region in synphilin-1. The indicated V5-synphilin-1 constructs were co-transfected with FLAG-kalirin-7. Synphilin-1 fragments were precipitated with anti-V5 antibodies. The precipitates were then probed with anti-FLAG antibodies to detect co-precipitated kalirin-7. The deletion mapping revealed that amino acids 1–348 of the synphilin-1 protein are crucial for the binding of kalirin-7. The asterisks indicate specific input signals of synphilin-1 fragments. For quantification of synphilin-1 fragment expression please refer to Figure S3 . (C) Endogenous synphilin-1 interacts with kalirin-7. Synphilin-1 was precipitated from whole-brain tissues (500 µg) of a wild type mouse with an anti-synphilin-1 antibody (Sigma). The precipitates were probed with a kalirin-7-specific antibody (KALRN from Abcam). Cell lysate of HEK293 cells overexpressed with FLAG-kalirin-7 and V5-synphilin-1 served as positive control. As a negative control brain lysate was subjected to immunoprecipitation without antibody. (D) Overlapping localization of kalirin-7 and synphilin-1 in cell culture. HEK293 cells were transiently transfected with both constructs for 6 h and stained with anti-FLAG and anti-V5 antibodies. The counterstaining was done with YoPro dye. The confocal sections demonstrate that both proteins display a punctate staining in the cytoplasm. Sph1, synphilin-1; Kal7, kalirin-7. Scale bar , 10 µm.

    Journal: PLoS ONE

    Article Title: The Guanine Nucleotide Exchange Factor Kalirin-7 Is a Novel Synphilin-1 Interacting Protein and Modifies Synphilin-1 Aggregate Transport and Formation

    doi: 10.1371/journal.pone.0051999

    Figure Lengend Snippet: Kalirin-7 interacts with synphilin-1 in vitro and in vivo . (A) Mapping of the interacting domain in the kalirin-7 protein. FLAG-kalirin-7 constructs as shown in the diagram were co-transfected with V5-synphilin-1 in HEK293 cells. 24 h after transfection, cells were subjected to immunoprecipitation with anti-FLAG agarose beads and subsequently kalirin-7 and synphilin-1 immunoreactivities were monitored applying anti-FLAG- or anti-V5 antibodies, respectively. IP indicates antibodies used for pulling down target proteins. IB indicates antibodies used for detection in western blot. The figure shows that kalirin-7 co-immunopreciptates with synphilin-1 and that spectrin repeats III and IV of the kalirin-7 protein are crucial for the interaction. Quantification of kalirin-7 fragment expression is shown in Figure S3 . (B) Mapping of the binding region in synphilin-1. The indicated V5-synphilin-1 constructs were co-transfected with FLAG-kalirin-7. Synphilin-1 fragments were precipitated with anti-V5 antibodies. The precipitates were then probed with anti-FLAG antibodies to detect co-precipitated kalirin-7. The deletion mapping revealed that amino acids 1–348 of the synphilin-1 protein are crucial for the binding of kalirin-7. The asterisks indicate specific input signals of synphilin-1 fragments. For quantification of synphilin-1 fragment expression please refer to Figure S3 . (C) Endogenous synphilin-1 interacts with kalirin-7. Synphilin-1 was precipitated from whole-brain tissues (500 µg) of a wild type mouse with an anti-synphilin-1 antibody (Sigma). The precipitates were probed with a kalirin-7-specific antibody (KALRN from Abcam). Cell lysate of HEK293 cells overexpressed with FLAG-kalirin-7 and V5-synphilin-1 served as positive control. As a negative control brain lysate was subjected to immunoprecipitation without antibody. (D) Overlapping localization of kalirin-7 and synphilin-1 in cell culture. HEK293 cells were transiently transfected with both constructs for 6 h and stained with anti-FLAG and anti-V5 antibodies. The counterstaining was done with YoPro dye. The confocal sections demonstrate that both proteins display a punctate staining in the cytoplasm. Sph1, synphilin-1; Kal7, kalirin-7. Scale bar , 10 µm.

    Article Snippet: Commercial antibodies used for western blotting (WB) analysis or immunofluorescence (IF) include rabbit anti-FLAG (WB, 1∶500; IF, 1∶200; Sigma), mouse anti-FLAG (IF, 1∶400; Sigma), mouse anti-V5 (WB, 1∶500; Sigma), rabbit anti-HA (WB, 1∶500; Sigma), rabbit anti-synphilin-1 (WB, 1∶1000; S5946, Sigma), goat anti-KALRN (WB, 1∶500; ab52012, Abcam), mouse anti-γ-tubulin (IF, 1∶100; clone GTU-88, Sigma), mouse anti-acetyl-tubulin (WB, 1∶4000; IF, 1∶200; clone 6-11-B1, Sigma), mouse anti-α-tubulin (WB, 1∶5000; clone B-5-1-2, Sigma), rabbit anti-ubiquitin (IF, 1∶300; Santa Cruz), Hsp27 (IF, 1∶200; C-20, Santa Cruz), and mouse anti-vimentin (IF, 1∶300; clone RV202, Abcam); all secondary antibodies were purchased from Amersham Biosciences.

    Techniques: In Vitro, In Vivo, Construct, Transfection, Immunoprecipitation, Western Blot, Expressing, Binding Assay, Positive Control, Negative Control, Cell Culture, Staining

    An HDAC6 deacetylase-dead mutant opposes the formation of synphilin-1 containing aggresomes mediated by kalirin-7. (A) HEK293 cells were triple-transfected with HcRed-synphilin-1, EGFP-kalirin-7 and FLAG-WT HDAC6 or FLAG-H216A/H611A mutant HDAC6. After 48 h cells were fixed and stained with anti-FLAG antibodies. Only cells co-expressing mutant HDAC6 form more cytoplasmic small aggregates (arrows). (B) Quantification shows that mutant HDAC6 inhibited the kalirin-7-mediated perinuclear synphilin-1 inclusion formation. The asterisks indicate statistical significance ( *** P ≤0.001). Error bars , S.E.

    Journal: PLoS ONE

    Article Title: The Guanine Nucleotide Exchange Factor Kalirin-7 Is a Novel Synphilin-1 Interacting Protein and Modifies Synphilin-1 Aggregate Transport and Formation

    doi: 10.1371/journal.pone.0051999

    Figure Lengend Snippet: An HDAC6 deacetylase-dead mutant opposes the formation of synphilin-1 containing aggresomes mediated by kalirin-7. (A) HEK293 cells were triple-transfected with HcRed-synphilin-1, EGFP-kalirin-7 and FLAG-WT HDAC6 or FLAG-H216A/H611A mutant HDAC6. After 48 h cells were fixed and stained with anti-FLAG antibodies. Only cells co-expressing mutant HDAC6 form more cytoplasmic small aggregates (arrows). (B) Quantification shows that mutant HDAC6 inhibited the kalirin-7-mediated perinuclear synphilin-1 inclusion formation. The asterisks indicate statistical significance ( *** P ≤0.001). Error bars , S.E.

    Article Snippet: Commercial antibodies used for western blotting (WB) analysis or immunofluorescence (IF) include rabbit anti-FLAG (WB, 1∶500; IF, 1∶200; Sigma), mouse anti-FLAG (IF, 1∶400; Sigma), mouse anti-V5 (WB, 1∶500; Sigma), rabbit anti-HA (WB, 1∶500; Sigma), rabbit anti-synphilin-1 (WB, 1∶1000; S5946, Sigma), goat anti-KALRN (WB, 1∶500; ab52012, Abcam), mouse anti-γ-tubulin (IF, 1∶100; clone GTU-88, Sigma), mouse anti-acetyl-tubulin (WB, 1∶4000; IF, 1∶200; clone 6-11-B1, Sigma), mouse anti-α-tubulin (WB, 1∶5000; clone B-5-1-2, Sigma), rabbit anti-ubiquitin (IF, 1∶300; Santa Cruz), Hsp27 (IF, 1∶200; C-20, Santa Cruz), and mouse anti-vimentin (IF, 1∶300; clone RV202, Abcam); all secondary antibodies were purchased from Amersham Biosciences.

    Techniques: Histone Deacetylase Assay, Mutagenesis, Transfection, Staining, Expressing

    Kalirin-7-mediated recruitment of synphilin-1 inclusions into aggresome is blocked by the HDAC inhibitor trichostatin A. (A) HEK293 cells expressing HcRed-synphilin-1 (a,b,c) or co-expressing HcRed-synphilin-1 and FLAG-kalirin-7 (d,e,f) were incubated in the presence of DMSO (a,d), 1 µM TSA (b,e) or 5 mM NaBu (c,f) for 18 h before being fixed and immunostained with anti-FLAG antibodies. The arrow indicates synphilin-1 cytoplasmic small aggregates. Blue , DAPI. Scale bar , 10 µm. (B) Quantification shows that treatment with the HDAC6 inhibitor TSA counteracts the recruitment of synphilin-1 into aggresomes mediated by kalirin-7, whereas the broad deacetylase inhibitor NaBu does not exert such an effect. The asterisks indicate statistical significance (** P ≤0.005). Error bars, S.E.

    Journal: PLoS ONE

    Article Title: The Guanine Nucleotide Exchange Factor Kalirin-7 Is a Novel Synphilin-1 Interacting Protein and Modifies Synphilin-1 Aggregate Transport and Formation

    doi: 10.1371/journal.pone.0051999

    Figure Lengend Snippet: Kalirin-7-mediated recruitment of synphilin-1 inclusions into aggresome is blocked by the HDAC inhibitor trichostatin A. (A) HEK293 cells expressing HcRed-synphilin-1 (a,b,c) or co-expressing HcRed-synphilin-1 and FLAG-kalirin-7 (d,e,f) were incubated in the presence of DMSO (a,d), 1 µM TSA (b,e) or 5 mM NaBu (c,f) for 18 h before being fixed and immunostained with anti-FLAG antibodies. The arrow indicates synphilin-1 cytoplasmic small aggregates. Blue , DAPI. Scale bar , 10 µm. (B) Quantification shows that treatment with the HDAC6 inhibitor TSA counteracts the recruitment of synphilin-1 into aggresomes mediated by kalirin-7, whereas the broad deacetylase inhibitor NaBu does not exert such an effect. The asterisks indicate statistical significance (** P ≤0.005). Error bars, S.E.

    Article Snippet: Commercial antibodies used for western blotting (WB) analysis or immunofluorescence (IF) include rabbit anti-FLAG (WB, 1∶500; IF, 1∶200; Sigma), mouse anti-FLAG (IF, 1∶400; Sigma), mouse anti-V5 (WB, 1∶500; Sigma), rabbit anti-HA (WB, 1∶500; Sigma), rabbit anti-synphilin-1 (WB, 1∶1000; S5946, Sigma), goat anti-KALRN (WB, 1∶500; ab52012, Abcam), mouse anti-γ-tubulin (IF, 1∶100; clone GTU-88, Sigma), mouse anti-acetyl-tubulin (WB, 1∶4000; IF, 1∶200; clone 6-11-B1, Sigma), mouse anti-α-tubulin (WB, 1∶5000; clone B-5-1-2, Sigma), rabbit anti-ubiquitin (IF, 1∶300; Santa Cruz), Hsp27 (IF, 1∶200; C-20, Santa Cruz), and mouse anti-vimentin (IF, 1∶300; clone RV202, Abcam); all secondary antibodies were purchased from Amersham Biosciences.

    Techniques: Expressing, Incubation, Histone Deacetylase Assay

    Kalirin-7 mediates perinuclear synphilin-1 inclusion formation in a microtubule-dependent manner. (A) HEK293 cells were cotransfected with HcRed-synphilin-1 and FLAG-kalirin-7. After 36 h cells were incubated with DMSO, 5 µM nocodazole or 10 µM colchicine for 12 h before being subjected to immunofluorescence with anti-FLAG and anti- β-tubulin antibodies. Cells expressing HcRed-synphilin-1 alone served as controls (arrowhead). In cells treated with nocodazole or colchicine, more cytoplasmic small aggregates (arrows) were formed. (B) Quantification (n > 250 cells per group) shows that nocodazole and colchicine inhibited the kalirin-7-mediated formation of synphilin-1-containing perinuclear inclusions. P, perinuclear aggregates; C, cytoplasmic small aggregates. The asterisks indicate statistical significance ( ** P ≤0.005). Error bars , S.E.

    Journal: PLoS ONE

    Article Title: The Guanine Nucleotide Exchange Factor Kalirin-7 Is a Novel Synphilin-1 Interacting Protein and Modifies Synphilin-1 Aggregate Transport and Formation

    doi: 10.1371/journal.pone.0051999

    Figure Lengend Snippet: Kalirin-7 mediates perinuclear synphilin-1 inclusion formation in a microtubule-dependent manner. (A) HEK293 cells were cotransfected with HcRed-synphilin-1 and FLAG-kalirin-7. After 36 h cells were incubated with DMSO, 5 µM nocodazole or 10 µM colchicine for 12 h before being subjected to immunofluorescence with anti-FLAG and anti- β-tubulin antibodies. Cells expressing HcRed-synphilin-1 alone served as controls (arrowhead). In cells treated with nocodazole or colchicine, more cytoplasmic small aggregates (arrows) were formed. (B) Quantification (n > 250 cells per group) shows that nocodazole and colchicine inhibited the kalirin-7-mediated formation of synphilin-1-containing perinuclear inclusions. P, perinuclear aggregates; C, cytoplasmic small aggregates. The asterisks indicate statistical significance ( ** P ≤0.005). Error bars , S.E.

    Article Snippet: Commercial antibodies used for western blotting (WB) analysis or immunofluorescence (IF) include rabbit anti-FLAG (WB, 1∶500; IF, 1∶200; Sigma), mouse anti-FLAG (IF, 1∶400; Sigma), mouse anti-V5 (WB, 1∶500; Sigma), rabbit anti-HA (WB, 1∶500; Sigma), rabbit anti-synphilin-1 (WB, 1∶1000; S5946, Sigma), goat anti-KALRN (WB, 1∶500; ab52012, Abcam), mouse anti-γ-tubulin (IF, 1∶100; clone GTU-88, Sigma), mouse anti-acetyl-tubulin (WB, 1∶4000; IF, 1∶200; clone 6-11-B1, Sigma), mouse anti-α-tubulin (WB, 1∶5000; clone B-5-1-2, Sigma), rabbit anti-ubiquitin (IF, 1∶300; Santa Cruz), Hsp27 (IF, 1∶200; C-20, Santa Cruz), and mouse anti-vimentin (IF, 1∶300; clone RV202, Abcam); all secondary antibodies were purchased from Amersham Biosciences.

    Techniques: Incubation, Immunofluorescence, Expressing

    Decreased tubulin acetylation in kalirin-7 expressing cells under TSA treatment. (A, B) An interaction of FLAG-kalirin-7, V5-synphilin-1 and HA-HDAC6 was examined by co-immunoprecipitation experiments. 24 h after transfection, HEK293 cells were lysed and 500 µg of protein lysates were subjected to immunoprecipitation with anti-FLAG or anti-V5 conjugated agarose beads, respectively. The precipitates were probed with HA antibodies to detect HDAC6 and revealed an interaction of HDAC6 with both kalirin-7 and synphilin-1. 30 µg of protein lysates were visualized as input control. The asterisk indicates a non-specific band observed in all raw lysates detected with anti-V5. (C) Cells transiently overexpressing FLAG-kalirin-7 (c, g), FLAG-kalirin-7 plus HcRed-synphilin-1 (d, h), or empty vectors (a, b, e, f) were immunostained for acetylated tubulin (a-d, green) and kalirin-7 (g, h light blue) after DMSO or 1 µM TSA treatment. While TSA treatment resulted in higher acetylation levels in comparison to controls (arrowheads), the overexpression of kalirin-7 led to a significant decrease of the α-tubulin acetylation levels (arrows). Blue, DAPI. Scale bar, 10 µm. (D) Acetylated tubulin levels were quantified by the fluorescence signal of individual cells, as described in Materials and Methods. Kalirin-7 transfected cells treated with TSA were compared to untransfected cells in the same cell population. Comparably, kalirin-7/synphilin-1 doubly transfected cells treated with TSA were quantified relative to untransfected cells in the same population. The asterisks indicate statistical significance ( ** P ≤0.005). Error bars , S.E., n = 100.

    Journal: PLoS ONE

    Article Title: The Guanine Nucleotide Exchange Factor Kalirin-7 Is a Novel Synphilin-1 Interacting Protein and Modifies Synphilin-1 Aggregate Transport and Formation

    doi: 10.1371/journal.pone.0051999

    Figure Lengend Snippet: Decreased tubulin acetylation in kalirin-7 expressing cells under TSA treatment. (A, B) An interaction of FLAG-kalirin-7, V5-synphilin-1 and HA-HDAC6 was examined by co-immunoprecipitation experiments. 24 h after transfection, HEK293 cells were lysed and 500 µg of protein lysates were subjected to immunoprecipitation with anti-FLAG or anti-V5 conjugated agarose beads, respectively. The precipitates were probed with HA antibodies to detect HDAC6 and revealed an interaction of HDAC6 with both kalirin-7 and synphilin-1. 30 µg of protein lysates were visualized as input control. The asterisk indicates a non-specific band observed in all raw lysates detected with anti-V5. (C) Cells transiently overexpressing FLAG-kalirin-7 (c, g), FLAG-kalirin-7 plus HcRed-synphilin-1 (d, h), or empty vectors (a, b, e, f) were immunostained for acetylated tubulin (a-d, green) and kalirin-7 (g, h light blue) after DMSO or 1 µM TSA treatment. While TSA treatment resulted in higher acetylation levels in comparison to controls (arrowheads), the overexpression of kalirin-7 led to a significant decrease of the α-tubulin acetylation levels (arrows). Blue, DAPI. Scale bar, 10 µm. (D) Acetylated tubulin levels were quantified by the fluorescence signal of individual cells, as described in Materials and Methods. Kalirin-7 transfected cells treated with TSA were compared to untransfected cells in the same cell population. Comparably, kalirin-7/synphilin-1 doubly transfected cells treated with TSA were quantified relative to untransfected cells in the same population. The asterisks indicate statistical significance ( ** P ≤0.005). Error bars , S.E., n = 100.

    Article Snippet: Commercial antibodies used for western blotting (WB) analysis or immunofluorescence (IF) include rabbit anti-FLAG (WB, 1∶500; IF, 1∶200; Sigma), mouse anti-FLAG (IF, 1∶400; Sigma), mouse anti-V5 (WB, 1∶500; Sigma), rabbit anti-HA (WB, 1∶500; Sigma), rabbit anti-synphilin-1 (WB, 1∶1000; S5946, Sigma), goat anti-KALRN (WB, 1∶500; ab52012, Abcam), mouse anti-γ-tubulin (IF, 1∶100; clone GTU-88, Sigma), mouse anti-acetyl-tubulin (WB, 1∶4000; IF, 1∶200; clone 6-11-B1, Sigma), mouse anti-α-tubulin (WB, 1∶5000; clone B-5-1-2, Sigma), rabbit anti-ubiquitin (IF, 1∶300; Santa Cruz), Hsp27 (IF, 1∶200; C-20, Santa Cruz), and mouse anti-vimentin (IF, 1∶300; clone RV202, Abcam); all secondary antibodies were purchased from Amersham Biosciences.

    Techniques: Expressing, Hemagglutination Assay, Immunoprecipitation, Transfection, Over Expression, Fluorescence

    Proposed pathway of kalirin-7-mediated synphilin-1 aggresome formation. (A) Under normal conditions, misfolded synphilin-1 is mainly accumulated in cytoplasmic small aggregates. (B) When kalirin-7 is overexpressed, it facilitates the recruitment of HDAC6 and the dynein motor complex and acts on microtubule dynamics by stimulating the deacetylase activity of HDAC6, thereby increasing the transportation of synphilin-1 into aggresomes.

    Journal: PLoS ONE

    Article Title: The Guanine Nucleotide Exchange Factor Kalirin-7 Is a Novel Synphilin-1 Interacting Protein and Modifies Synphilin-1 Aggregate Transport and Formation

    doi: 10.1371/journal.pone.0051999

    Figure Lengend Snippet: Proposed pathway of kalirin-7-mediated synphilin-1 aggresome formation. (A) Under normal conditions, misfolded synphilin-1 is mainly accumulated in cytoplasmic small aggregates. (B) When kalirin-7 is overexpressed, it facilitates the recruitment of HDAC6 and the dynein motor complex and acts on microtubule dynamics by stimulating the deacetylase activity of HDAC6, thereby increasing the transportation of synphilin-1 into aggresomes.

    Article Snippet: Commercial antibodies used for western blotting (WB) analysis or immunofluorescence (IF) include rabbit anti-FLAG (WB, 1∶500; IF, 1∶200; Sigma), mouse anti-FLAG (IF, 1∶400; Sigma), mouse anti-V5 (WB, 1∶500; Sigma), rabbit anti-HA (WB, 1∶500; Sigma), rabbit anti-synphilin-1 (WB, 1∶1000; S5946, Sigma), goat anti-KALRN (WB, 1∶500; ab52012, Abcam), mouse anti-γ-tubulin (IF, 1∶100; clone GTU-88, Sigma), mouse anti-acetyl-tubulin (WB, 1∶4000; IF, 1∶200; clone 6-11-B1, Sigma), mouse anti-α-tubulin (WB, 1∶5000; clone B-5-1-2, Sigma), rabbit anti-ubiquitin (IF, 1∶300; Santa Cruz), Hsp27 (IF, 1∶200; C-20, Santa Cruz), and mouse anti-vimentin (IF, 1∶300; clone RV202, Abcam); all secondary antibodies were purchased from Amersham Biosciences.

    Techniques: Histone Deacetylase Assay, Activity Assay

    Kalirin-7 decreases synphilin-1-induced aggregates in biochemical and live cell analysis. (A) HEK293 cells were transfected with HcRed-synphilin-1 alone or cotransfected with FLAG-kalirin-7. HcRed empty vector served as control. Cells were lysed 24, 48 or 72 h after transfection, fractionated by AGERA on 2% agarose gels and analyzed by western blotting with an antibody recognizing synphilin-1 aggregates. S, HcRed-synphilin-1; K, FLAG-kalirin-7; C, control (HcRed empty vector). Indicated by a bracket on the right is the the major area of aggregate signal which was used for quantification. (B) Quantification of AGERA blots of 3 independent experiments for each time point and condition relative to the mean expression level of controls at 24 hrs post-transfection confirmed an increase of aggregates over time and a reduced number of aggregates in cells doubly transfected with kalirin-7 and synphilin-1 compared to cells transfected with synphilin-1 alone at 48 and 72 hours. (C) Long-term time-lapse imaging. HEK293 cells were transfected with HcRed-synphilin-1 and empty EGFP vector (upper chart) or EGFP-kalirin-7 (lower chart) and observed by live cell imaging fluorescent microscopy (Cell Observer, equipped with an Axio Observer.Z1 and an ApoTome Imaging System Zeiss, Germany) at 37°C. Depicted are average intensity projections of 6–8 ApoTome optical slides encompassing the entire height of the cells. Time-points indicate hours post-transfection. Images were merged from red, green and phase contrast channels. Arrows indicate the cell traced over the experimental time. Scale bar , 10 µm. (D) Quantification (n > 35 cells per group) of the time-lapse imaging shows that aggregate numbers are reduced when FLAG-kalirin-7 is coexpressed. Light gray bars: Sph alone; dark gray bars: Sph and Kal7 coexpression. Results represent the average of three independent experiments. The asterisks indicate statistical significance (* P ≤0.05). Error bars , S.E.

    Journal: PLoS ONE

    Article Title: The Guanine Nucleotide Exchange Factor Kalirin-7 Is a Novel Synphilin-1 Interacting Protein and Modifies Synphilin-1 Aggregate Transport and Formation

    doi: 10.1371/journal.pone.0051999

    Figure Lengend Snippet: Kalirin-7 decreases synphilin-1-induced aggregates in biochemical and live cell analysis. (A) HEK293 cells were transfected with HcRed-synphilin-1 alone or cotransfected with FLAG-kalirin-7. HcRed empty vector served as control. Cells were lysed 24, 48 or 72 h after transfection, fractionated by AGERA on 2% agarose gels and analyzed by western blotting with an antibody recognizing synphilin-1 aggregates. S, HcRed-synphilin-1; K, FLAG-kalirin-7; C, control (HcRed empty vector). Indicated by a bracket on the right is the the major area of aggregate signal which was used for quantification. (B) Quantification of AGERA blots of 3 independent experiments for each time point and condition relative to the mean expression level of controls at 24 hrs post-transfection confirmed an increase of aggregates over time and a reduced number of aggregates in cells doubly transfected with kalirin-7 and synphilin-1 compared to cells transfected with synphilin-1 alone at 48 and 72 hours. (C) Long-term time-lapse imaging. HEK293 cells were transfected with HcRed-synphilin-1 and empty EGFP vector (upper chart) or EGFP-kalirin-7 (lower chart) and observed by live cell imaging fluorescent microscopy (Cell Observer, equipped with an Axio Observer.Z1 and an ApoTome Imaging System Zeiss, Germany) at 37°C. Depicted are average intensity projections of 6–8 ApoTome optical slides encompassing the entire height of the cells. Time-points indicate hours post-transfection. Images were merged from red, green and phase contrast channels. Arrows indicate the cell traced over the experimental time. Scale bar , 10 µm. (D) Quantification (n > 35 cells per group) of the time-lapse imaging shows that aggregate numbers are reduced when FLAG-kalirin-7 is coexpressed. Light gray bars: Sph alone; dark gray bars: Sph and Kal7 coexpression. Results represent the average of three independent experiments. The asterisks indicate statistical significance (* P ≤0.05). Error bars , S.E.

    Article Snippet: Commercial antibodies used for western blotting (WB) analysis or immunofluorescence (IF) include rabbit anti-FLAG (WB, 1∶500; IF, 1∶200; Sigma), mouse anti-FLAG (IF, 1∶400; Sigma), mouse anti-V5 (WB, 1∶500; Sigma), rabbit anti-HA (WB, 1∶500; Sigma), rabbit anti-synphilin-1 (WB, 1∶1000; S5946, Sigma), goat anti-KALRN (WB, 1∶500; ab52012, Abcam), mouse anti-γ-tubulin (IF, 1∶100; clone GTU-88, Sigma), mouse anti-acetyl-tubulin (WB, 1∶4000; IF, 1∶200; clone 6-11-B1, Sigma), mouse anti-α-tubulin (WB, 1∶5000; clone B-5-1-2, Sigma), rabbit anti-ubiquitin (IF, 1∶300; Santa Cruz), Hsp27 (IF, 1∶200; C-20, Santa Cruz), and mouse anti-vimentin (IF, 1∶300; clone RV202, Abcam); all secondary antibodies were purchased from Amersham Biosciences.

    Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Imaging, Live Cell Imaging, Microscopy

    Kalirin-7 alters synphilin-1-induced inclusion formation. (A) When HEK293 cells were transfected with HcRed-synphilin-1 alone (a,b), FLAG-kalirin-7 (c) or both expression constructs (d) for 48 h, two types of inclusions were observed: small cytoplasmic aggregates (arrowhead; a) and perinuclear aggregates (arrow; b, d). Blue , DAPI. Scale bar , 10 µm. (B) Quantitative analysis of the experiment described in (A). HcRed-synphilin-1 was expressed without or with FLAG-kalirin-7 for 48 h. Cells were fixed and immunostained with anti-FLAG antibodies. Cells with cytoplasmic small aggregates, perinuclear aggregates or soluble synphilin-1 were counted. Results represent the average of three independent experiments. (C) Total numbers of aggregates per cell (cytoplasmic and perinuclear) were counted applying ApoTome confocal fluorescent microscopy. Over 100 cells were counted for each condition. The asterisks indicate statistical significance (**P≤0.005; ***P≤0.001). Error bars , S.E.

    Journal: PLoS ONE

    Article Title: The Guanine Nucleotide Exchange Factor Kalirin-7 Is a Novel Synphilin-1 Interacting Protein and Modifies Synphilin-1 Aggregate Transport and Formation

    doi: 10.1371/journal.pone.0051999

    Figure Lengend Snippet: Kalirin-7 alters synphilin-1-induced inclusion formation. (A) When HEK293 cells were transfected with HcRed-synphilin-1 alone (a,b), FLAG-kalirin-7 (c) or both expression constructs (d) for 48 h, two types of inclusions were observed: small cytoplasmic aggregates (arrowhead; a) and perinuclear aggregates (arrow; b, d). Blue , DAPI. Scale bar , 10 µm. (B) Quantitative analysis of the experiment described in (A). HcRed-synphilin-1 was expressed without or with FLAG-kalirin-7 for 48 h. Cells were fixed and immunostained with anti-FLAG antibodies. Cells with cytoplasmic small aggregates, perinuclear aggregates or soluble synphilin-1 were counted. Results represent the average of three independent experiments. (C) Total numbers of aggregates per cell (cytoplasmic and perinuclear) were counted applying ApoTome confocal fluorescent microscopy. Over 100 cells were counted for each condition. The asterisks indicate statistical significance (**P≤0.005; ***P≤0.001). Error bars , S.E.

    Article Snippet: Commercial antibodies used for western blotting (WB) analysis or immunofluorescence (IF) include rabbit anti-FLAG (WB, 1∶500; IF, 1∶200; Sigma), mouse anti-FLAG (IF, 1∶400; Sigma), mouse anti-V5 (WB, 1∶500; Sigma), rabbit anti-HA (WB, 1∶500; Sigma), rabbit anti-synphilin-1 (WB, 1∶1000; S5946, Sigma), goat anti-KALRN (WB, 1∶500; ab52012, Abcam), mouse anti-γ-tubulin (IF, 1∶100; clone GTU-88, Sigma), mouse anti-acetyl-tubulin (WB, 1∶4000; IF, 1∶200; clone 6-11-B1, Sigma), mouse anti-α-tubulin (WB, 1∶5000; clone B-5-1-2, Sigma), rabbit anti-ubiquitin (IF, 1∶300; Santa Cruz), Hsp27 (IF, 1∶200; C-20, Santa Cruz), and mouse anti-vimentin (IF, 1∶300; clone RV202, Abcam); all secondary antibodies were purchased from Amersham Biosciences.

    Techniques: Transfection, Expressing, Construct, Microscopy

    Characterization of synphilin-1-containing aggregates as aggresomes. HEK293 cells coexpressing HcRed-synphilin-1 and FLAG-kalirin-7 were fixed 48 h post-transfection and subsequently stained with the indicated antibodies. Arrows indicate the colocalization between synphilin-1 inclusions and γ-tubulin, ubiquitin and Hsp27 while the intermediate filament protein vimentin forms a cage surrounding a pericentriolar core of aggregates. Merged images are shown to the right. Blue , DAPI. Scale bar , 10 µm.

    Journal: PLoS ONE

    Article Title: The Guanine Nucleotide Exchange Factor Kalirin-7 Is a Novel Synphilin-1 Interacting Protein and Modifies Synphilin-1 Aggregate Transport and Formation

    doi: 10.1371/journal.pone.0051999

    Figure Lengend Snippet: Characterization of synphilin-1-containing aggregates as aggresomes. HEK293 cells coexpressing HcRed-synphilin-1 and FLAG-kalirin-7 were fixed 48 h post-transfection and subsequently stained with the indicated antibodies. Arrows indicate the colocalization between synphilin-1 inclusions and γ-tubulin, ubiquitin and Hsp27 while the intermediate filament protein vimentin forms a cage surrounding a pericentriolar core of aggregates. Merged images are shown to the right. Blue , DAPI. Scale bar , 10 µm.

    Article Snippet: Commercial antibodies used for western blotting (WB) analysis or immunofluorescence (IF) include rabbit anti-FLAG (WB, 1∶500; IF, 1∶200; Sigma), mouse anti-FLAG (IF, 1∶400; Sigma), mouse anti-V5 (WB, 1∶500; Sigma), rabbit anti-HA (WB, 1∶500; Sigma), rabbit anti-synphilin-1 (WB, 1∶1000; S5946, Sigma), goat anti-KALRN (WB, 1∶500; ab52012, Abcam), mouse anti-γ-tubulin (IF, 1∶100; clone GTU-88, Sigma), mouse anti-acetyl-tubulin (WB, 1∶4000; IF, 1∶200; clone 6-11-B1, Sigma), mouse anti-α-tubulin (WB, 1∶5000; clone B-5-1-2, Sigma), rabbit anti-ubiquitin (IF, 1∶300; Santa Cruz), Hsp27 (IF, 1∶200; C-20, Santa Cruz), and mouse anti-vimentin (IF, 1∶300; clone RV202, Abcam); all secondary antibodies were purchased from Amersham Biosciences.

    Techniques: Transfection, Staining