Journal: The Journal of Neuroscience

Article Title: Microglial mTOR is Neuronal Protective and Antiepileptogenic in the Pilocarpine Model of Temporal Lobe Epilepsy

doi: 10.1523/JNEUROSCI.2754-19.2020

Figure Lengend Snippet: Key resources, related to experimental procedures

Article Snippet: Rat anti-mouse CD68 , Bio-Rad , Catalog #MCA1957; RRID: AB_322219.

Techniques: Recombinant, In Situ, cDNA Synthesis, SYBR Green Assay, Software

Journal: The Journal of Neuroscience

Article Title: Microglial mTOR is Neuronal Protective and Antiepileptogenic in the Pilocarpine Model of Temporal Lobe Epilepsy

doi: 10.1523/JNEUROSCI.2754-19.2020

Figure Lengend Snippet: Activation of mTOR in microglia following SE. A, Confocal images acquired from CTX, hippocampus CA1 and CA3, and the DG. Scale bar, 20 µm. B, C, Quantification of p-S6/Iba1 double-positive microglial cells (B) and CD68/Iba1 double-positive microglial cells (C) in sham-treated (n = 4; 2 males and 2 females), SE-3d (n = 5; 2 males and 3 females), and SE-3d+rapamycin (n = 4; 2 males and 2 females) mice. Data are presented as the mean ± SEM. Sample comparison was made among three groups (sham, SE, and SE + rapamycin groups) and analyzed with one-way ANOVA (P1), followed by Tukey's multiple-comparisons test (P2, comparison between control and SE; P3, comparison between SE and SE + rapamycin).

Article Snippet: Rat anti-mouse CD68 , Bio-Rad , Catalog #MCA1957; RRID: AB_322219.

Techniques: Activation Assay, Comparison, Control

Journal: The Journal of Neuroscience

Article Title: Microglial mTOR is Neuronal Protective and Antiepileptogenic in the Pilocarpine Model of Temporal Lobe Epilepsy

doi: 10.1523/JNEUROSCI.2754-19.2020

Figure Lengend Snippet: Microglial proliferation and activation in control and mTORCx3cr1-creCKO mice following SE. A, Full montage of confocal images showing acute injury (FJB+, green), microglial density (Iba1+, green), and CD68 expression (CD68+, red) in the hippocampus of control and mTORCx3cr1-creCKO mice either sham-treated or 1–7 d post-SE. Scale bar, 200 µm. B, Representative confocal images acquired from the CA1 of control and mTORCx3cr1-creCKO mice either sham treated or 1–7 d post-SE. Scale bar, 20 µm. C, Representative confocal images showing Ki67 expression (Ki67+, red) in the CA1 of control and mTORCx3cr1-creCKO mice either sham treated or 1–7 d post-SE. Scale bar, 20 µm. D, Average doses of pilocarpine that induced stage 4/5 seizures in control and mTORCx3cr1-creCKO mice. E, F, Quantification of Iba1+ microglia and CD68 immunofluorescence intensity in control and mTORCx3cr1-creCKO mice either sham-treated or 1-7 d post-SE. Sample sizes in control groups: sham (n = 6; 3 males and 3 females), SE-1d (n = 6; 3 males and 3 females), SE-3d (n = 6; 3 males and 3 females), and SE-7d (n = 6; 3 males and 3 females); and in the mTORCx3cr1-creCKO groups: sham (n = 4; 2 males and 2 females), SE-1d (n = 5; 2 males and 3 females), SE3d (n = 6; 3 males and 3 females), and SE-7d (n = 4; 2 males and 2 females). G, Quantification of Ki67/Iba1 double-positive microglial cells in control and mTORCx3cr1-creCKO mice either sham treated or 1–7 d post-SE. Sample sizes in control groups: sham (n = 6; 3 males and 3 females), SE-1d (n = 6; 3 males and 3 females), SE-3d (n = 6; 3 males and 3 females), and SE-7d (n = 6; 3 males and 3 females); and in the mTORCx3cr1-creCKO groups: sham (n = 4; 2 males and 2 females), SE-1d (n = 5; 2 males and 3 females), SE3d (n = 6; 3 males and 3 females), and SE-7d (n = 4; 2 males and 2 females). Data are presented as the mean ± SEM A two-tailed unpaired t test was used for the comparison between control and mTORCx3cr1-creCKO mice.

Article Snippet: Rat anti-mouse CD68 , Bio-Rad , Catalog #MCA1957; RRID: AB_322219.

Techniques: Activation Assay, Control, Expressing, Immunofluorescence, Two Tailed Test, Comparison

Journal: The Journal of Neuroscience

Article Title: Wnt5a Is a Transcriptional Target of Dlx Homeogenes and Promotes Differentiation of Interneuron Progenitors In Vitro and In Vivo

doi: 10.1523/JNEUROSCI.3110-10.2011

Figure Lengend Snippet: Expression of Wnt5a and Wnt5b in normal and Dlx5−/− OB. A, B, ISH on coronal sections of E14.5 (A) and neonatal (B) OB of WT and Dlx5−/− specimens (genotypes are reported on top). Expression of Wnt5b (a, b, a′, b′), Wnt5a (c, d, c′, d′), Dlx2 (e, f, e′, f′), Dlx5 (g, g′), and Dlx5-lacZ (X-gal staining) (h) is shown. In the WT embryonic OB, Wnt5b is expressed in VZ–SVZ region; conversely, Wnt5a is not expressed in this region and instead is expressed in a region overlapping with both Dlx2 and Dlx5 in the ventral–medial OB. In the neonatal brain (left), coexpression of Wnt5a, Dlx2, and Dlx5 is observed in the differentiated layers of the OB. The expression of Wnt5a is reduced in the Dlx5−/− specimens at both ages (black arrows). C, ISH for Wnt5a (left) or Wnt5b (right) on coronal sections of E14.5 OB from Dlx5-lacZ+/− embryos (blue signal), followed by immunostaining for β-gal (brown signal). Lower (i, i′) and higher (j, j′) magnification micrographs are shown. D, Diagram illustrating the overlapping expression of Wnt5a (green bar), Wnt5b (yellow bar), Dlx2 (red bar), and Dlx5 (blue bar) in the embryonic OB, and their position relative to the VZ–SVZ. The layers and neurons indicated in the scheme refer to the situation at P0. GCL, Granule cell layer; GL, glomerular layer; LV, lateral ventricle; MCL, mitral/tufted cell layer; ONL, olfactory nerve layer. E, Real-time qPCR analyses on RNA extracted from WT and Dlx5−/− OBs at E14.5 (left) and at birth (right). Reduced expression of Wnt5a, but not Wnt5b, is observed in both cases. Samples are from pools of tissues (at least 3). Error bars represent the mean ± SD of two independent experiments. Differences were statistically significant as measured by a two-tailed Student's t test (*p < 0.001). Scale bars: 400 μm (a–h, i, i′), 200 μm (a′–g′), 100 μm (j, j′).

Article Snippet: The DLX5-myc and DLX2-myc expression vectors (OriGene) containing, respectively, the full-length human DLX5 and DLX2 cDNA with an in-frame insertion of the myc-TAG at the C terminus, were used.

Techniques: Expressing, Staining, Immunostaining, Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: Wnt5a Is a Transcriptional Target of Dlx Homeogenes and Promotes Differentiation of Interneuron Progenitors In Vitro and In Vivo

doi: 10.1523/JNEUROSCI.3110-10.2011

Figure Lengend Snippet: Expression of Dlx, Wnt5a, and differentiation markers in NS cells. A, Real-time qPCR analysis for the expression of Nestin, Dlx2, Dlx5, GAD1, GAD2, CR, and Wnt5a mRNA in NS cells, at two time points (T4 and T10, indicating 4 and 10 d in vitro) following the application of a differentiation stimulus (see Materials and Methods). T0 corresponds to proliferating cells. Dlx2 is expressed in proliferating cells, while Dlx5 is upregulated during differentiation, followed by GAD-1, GAD-2, CR, and Wnt5a. One “referee” sample was introduced in every experiment, and used for normalization. Error bars represent the mean ± SD of three independent experiments. Differences were statistically significant as measured by a two-tailed Student's t test (p < 0.05). B, Top row, Immunostaining on proliferating NS cells (maintained in EGF + FGF2) to show absence of pan-Dlx staining (a), CR (b), and GABA (c). Middle row, Immunostaining on differentiating cells (10 DIV) showing expression of pan-Dlx proteins (d), CR (e), and GABA (f). Bottom row, Double immunostaining to confirm the coexpression of Dlx proteins (nuclear staining) with CR (g), GABA (h), and GAD67 (i). Few GAD67+/Dlx− cells were observed in these cultures (h′). Scale bar (in a), 50 μm.

Article Snippet: The DLX5-myc and DLX2-myc expression vectors (OriGene) containing, respectively, the full-length human DLX5 and DLX2 cDNA with an in-frame insertion of the myc-TAG at the C terminus, were used.

Techniques: Expressing, In Vitro, Two Tailed Test, Immunostaining, Staining, Double Immunostaining

Journal: The Journal of Neuroscience

Article Title: Wnt5a Is a Transcriptional Target of Dlx Homeogenes and Promotes Differentiation of Interneuron Progenitors In Vitro and In Vivo

doi: 10.1523/JNEUROSCI.3110-10.2011

Figure Lengend Snippet: Dlx proteins regulate Wnt5a transcription and bind to the Wnt5a locus in native chromatin. A, NS cells nucleofected with EGFP (a), Dlx2-mycTAG (b), or Dlx5-mycTAG (c) expression vector, stained with anti-mycTAG antibody (green arrows). Scale bar (in a), 100 μm. B, Western blot analysis on total proteins from NS cells nucleofected with Dlx2-mycTAG, Dlx5-mycTAG, or the empty vector (mock), revealed with anti-mycTAG antibody. C, Luc-reporter transcription activity of Dlx2- and Dlx5-mycTAG proteins on the Dlx-responsive element of Arx. Experiments were done by nucleofecting NS cells. Values are expressed as relative Luc activity (RLU), normalized against mock nucleofection and Renilla expression. Mean values of three experiments are shown. D, Top, Diagram of the Wnt5a locus to report the position and sequence of the putative homeodomain binding sites in the A1, A2, and A3 fragments. Bottom, Luc transactivation activity of Dlx2- and Dlx5-mycTAG proteins on the A1, A2, and A3 fragments of the Wnt5a locus, inserted in combination with the Wnt5a promoter. Error bars represent the mean ± SD of three independent experiments. Differences were statistically significant as measured by a two-tailed Student's t test (*p < 0.05). E, ChIP analysis on chromatin from the ST14A cells transfected with the Dlx2- and the Dlx5-mycTAG vectors, precipitated with anti-mycTAG antibody, followed by PCR analysis with primers for fragments in the A2 and A3 regions of the Wnt5a locus. As negative control, cells were transfected with the empty vector, and the ChIP was performed with anti-IgG alone. As input, unprecipitated chromatin was used for PCR amplification. The signals were quantified by digital densitometry using the Quantity One software (version 4.5.2). Enrichment is observed upon transfection with both Dlx2-myc and Dlx5-myc with both primers used. Quantification is reported on the histograms on the right; values were normalized against empty vector. Errors bars represent the mean ± SD of two independent experiments.

Article Snippet: The DLX5-myc and DLX2-myc expression vectors (OriGene) containing, respectively, the full-length human DLX5 and DLX2 cDNA with an in-frame insertion of the myc-TAG at the C terminus, were used.

Techniques: Expressing, Plasmid Preparation, Staining, Western Blot, Activity Assay, Sequencing, Binding Assay, Two Tailed Test, Transfection, Negative Control, Amplification, Software

Journal: The Journal of Neuroscience

Article Title: Wnt5a Is a Transcriptional Target of Dlx Homeogenes and Promotes Differentiation of Interneuron Progenitors In Vitro and In Vivo

doi: 10.1523/JNEUROSCI.3110-10.2011

Figure Lengend Snippet: Model of regulation of GABA+ neuron differentiation in the olfactory bulb by Dlx and Wnt5a. The activity of canonical (represented by Wnt3a) and noncanonical (Wnt5a) Wnt signaling is regionally and functionally separated (indicated on the top). Transcriptional regulations are represented by solid arrows, and ligand–receptor interactions or other non-cell-autonomous regulations are represented by dashed arrows. Dlx2 is generally considered transcriptionally upstream of Dlx5 and Dlx6, but our data suggest that it may also regulate transcription of Wnt5a.

Article Snippet: The DLX5-myc and DLX2-myc expression vectors (OriGene) containing, respectively, the full-length human DLX5 and DLX2 cDNA with an in-frame insertion of the myc-TAG at the C terminus, were used.

Techniques: Activity Assay

Journal: Cell Death and Differentiation

Article Title: FOXQ1 controls the induced differentiation of melanocytic cells

doi: 10.1038/s41418-018-0066-y

Figure Lengend Snippet: FOXQ1 regulates cAMP/CREB1-dependent pigmentation. a NHM were treated with the indicated doses of Forskolin (FSK) for 5 h (left panel) or with 50 μM of FSK for indicated duration (right panel) followed by immunoblotting with the indicated antibodies. b NHM were transduced with control (CL) or FOXQ1 (F1, F2) shRNAs and treated with vehicle (DMSO) or the indicated doses of Forskolin (FSK) for 5 h followed by immunoblotting with the indicated antibodies. c Melanin content in cells described in b. d Representative images of ears from Foxq1+/+ or Foxq1–/– mice (n = 3) treated with either vehicle (DMSO, left ear) or vehicle containing 100μM Forskolin (FSK, right ear). e Pigmentation was quantified using a reflectance spectrometer and represented as fold change in the coefficient of absorption “mua”. f Representative images of H&E staining of mouse ear tissues shown in d. All data represent mean ± SEM. Statistical significance was assessed using two-tailed Student’s t-tests. A p < 0.05 (*) was considered significant

Article Snippet: CREB1 cDNA was purchased from DNASU repository (DNASU, HsCD00441528). shRNA targeting CREB1 was purchased from OriGene (RC210577). shRNAs targeting human and mouse FOXQ1, MITF, pLV-puro, and pLKO-1.puro lentiviral control vectors were purchased from Sigma-Aldrich (St. Louis, MO). pLKO.1 puro shRNA β-catenin was a gift from Bob Weinberg (Addgene plasmid #18803). pLenti-CMV-Puro-LUC was purchased from Addgene (w168-1-7477).

Techniques: Western Blot, Transduction, Staining, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: FOXQ1 controls the induced differentiation of melanocytic cells

doi: 10.1038/s41418-018-0066-y

Figure Lengend Snippet: FOXQ1 is important for cAMP/CREB1-dependent pigmentation. a, b Cells transduced with control shRNA (CL) or CREB1 shRNA (C1) were probed in immunoblotting with the indicated antibodies or in Q-RT-PCR. CREB1/Actb, Foxq1/Actb, and Mitf/Actb signal ratios are shown. c Melanin content and representative cell pellet images of cells described in a, b. d Schematic representation of human and mouse FOXQ1 promoter. Diamonds represent potential CREB1 consensus binding sites. e NHM, Melan-a, and B16 cells treated with vehicle (DMSO) or 10 μM Forskolin were probed in ChIP assay. Shown are ratios of Q-PCR signals in reactions with DNA precipitated with CREB1 or IgG antibodies using primers corresponding to potential CREB1-binding sites in FOXQ1 promoter. NS corresponds to CREB1-nonspecific primer targeting a distal genomic region. All data represent mean ± SEM. Statistical significance was assessed using two-tailed Student’s t-tests. A p < 0.05 (*) was considered significant

Article Snippet: CREB1 cDNA was purchased from DNASU repository (DNASU, HsCD00441528). shRNA targeting CREB1 was purchased from OriGene (RC210577). shRNAs targeting human and mouse FOXQ1, MITF, pLV-puro, and pLKO-1.puro lentiviral control vectors were purchased from Sigma-Aldrich (St. Louis, MO). pLKO.1 puro shRNA β-catenin was a gift from Bob Weinberg (Addgene plasmid #18803). pLenti-CMV-Puro-LUC was purchased from Addgene (w168-1-7477).

Techniques: Transduction, shRNA, Western Blot, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Two Tailed Test

Journal: Cell reports

Article Title: FOS licenses early events in stem cell activation driving skeletal muscle regeneration

doi: 10.1016/j.celrep.2020.108656

Figure Lengend Snippet: (A) Workflow for isolating fresh SCs (I) and single fibers (II) from skeletal muscle and time points included in the in vivo injury time course (III). (B) Dot plots showing analysis for (II) GFP expression in CD45 − /CD11b − /Ter119 − /CD31 − /Sca1 − /β1-Integrin + /CXCR4 + fresh SCs (I) from uninjured muscles of wild-type (FMO-WT, top) or Fos GFP (bottom) mice. SSC-A, side scatter area. Data were pre-gated on physical and live cell parameters (see for details). (C) Mean (±SD) percentage of fresh Fos GFP SCs expressing GFP (compiled analysis from 15 mice). (D) Pre-fixed (bottom) or non-pre-fixed (standard isolation, top) single fibers co-stained for PAX7 (green), FOS (red), and DAPI (blue). (E) Quantification (mean ± SD) of the percentage of PAX7+ SCs expressing FOS in freshly isolated single fibers stained as in (D). Data represent enumeration of more than 100 SCs across a minimum of 30 fibers per biological replicate for each condition (n = 3 mice per condition). (F) Fresh-frozen muscle sections co-stained for PAX7 (green), FOS (red), and DAPI (blue) 0, 1.5, and 12 h after cardiotoxin (CTX; 10 μM) injury. All channels are shown separately for the 1.5 h post-injury field (bottom row) and merged for 0, 1.5, and 12 h (top row). (G and H) Quantification of immunofluorescence (IF) data shown in (F), including (G) mean (±SD) percentage of PAX7+ SCs expressing FOS protein and (H) mean (±SD) number of PAX7+ SCs quantified per TA/ extensor digitorum longus (EDL) section per condition (n = 3 mice per time point). Student’s two-tailed unpaired t test (E) and one-way ANOVA with Tukey post hoc test (G and H). The scale bars represent 50 mm (D) and 100 μm (F). See also .

Article Snippet: Mouse anti-mouse PAX7 , DSHB , RRID: AB_528428.

Techniques: In Vivo, Expressing, Muscles, Isolation, Staining, Immunofluorescence, Two Tailed Test

Journal: Cell reports

Article Title: FOS licenses early events in stem cell activation driving skeletal muscle regeneration

doi: 10.1016/j.celrep.2020.108656

Figure Lengend Snippet: (A) Schematic showing the experimental design and FACS gating strategy for isolation of 1,000 Fresh Fos GFP+ and 1,000 Fos GFP− SCs directly sorted for RNA-seq analysis (SCs isolated from 4 mice). (B) Hierarchically clustered heatmap showing all 3,387 differentially expressed genes (DEGs; >1.5 FC, FDR < 0.05) in Fos GFP+ versus Fos GFP− SCs. (C) Volcano plot highlighting known SC regulator genes enriched (blue) or depleted (red) in fresh Fos GFP+ SCs. Notable mRNAs not significantly changed are indicated in black. (D) Top ranked Biocarta pathways associated with enriched genes in Fos GFP+ fresh SCs. (E) Venn diagrams showing overlap in genes enriched in Fos GFP+ or Fos GFP− SCs and in T3 (standard isolation, top) or T0 ( in-situ-fixed , quiescent SCs, bottom) SCs, respectively . The p values were determined by Fisher’s exact test of significance. (F) Heatmap of MAPK targets expressed in Fos GFP+ SCs relative to Fos GFP SCs. (G) Strategy for testing whether p38 MAPK induces FOS in freshly isolated single fibers. (H) Single fibers co-stained for PAX7 and FOS after isolation in the presence of vehicle or the p38 MAPK inhibitor SB202190 (SB). Scale bar, 50 μm. (I) Mean (±SD) percentage of PAX7+ SCs expressing FOS protein after isolation under the indicated condition. Data represent enumeration of more than 100 SCs across a minimum of 30 fibers per biological replicate for each condition (n = 3 mice per condition). The Z score equals the number of SDs from the mean expression of all genes (C and F). Fisher’s exact test (E) and Student’s two-tailed unpaired t test (I). See also .

Article Snippet: Mouse anti-mouse PAX7 , DSHB , RRID: AB_528428.

Techniques: Isolation, RNA Sequencing, In Situ, Staining, Expressing, Two Tailed Test

Journal: Cell reports

Article Title: FOS licenses early events in stem cell activation driving skeletal muscle regeneration

doi: 10.1016/j.celrep.2020.108656

Figure Lengend Snippet: (A) 1,500 Fos cKO or 1,500 fresh control SCs were cultured for 7 days in GM. Shown is quantification of the mean (±SD) number of Hoechst+ cells per well (n = 3 mice per genotype). (B) 2,000 fresh Fos cKO or 2,000 control SCs cultured in GM for 4 or 7 days and pulsed with EdU 3 h before harvest, displaying EdU-positive (magenta) and Hoechst-positive (blue) nuclei and mean (±SD) percentage of EdU+ nuclei among total Hoechst+ cells after 4 (n = 2–4 mice per genotype) or 7 days (n = 3 mice per genotype) in culture. (C) Schematic showing the tamoxifen (TAM) treatment regimen before and after a freeze muscle injury (cryoinjury) in the TA muscle. (D) Representative Laminin-stained Fos cKO and control muscle sections (20×) from uninjured mice (top) and from mice 7 (center) and 50 (bottom) days after freeze injury. (E and F) Distribution (E) and mean (±SD) cross-sectional area (CSA; F) of fiber sizes from uninjured Fos cKO or control animals (n = 4 mice per genotype). (G and H) Distribution (G) and mean (±SD) CSA (H) of regenerating (centrally nucleated) muscle fibers 7 days after freeze injury (n = 5 mice per genotype). (I) Quantification of the total number of muscle fibers per TA/EDL section in control and Fos cKO mice before (left) and 50 days after freeze injury (right) (n = 5 mice per genotype per condition). (J) Total number of Pax7+ SCs in uninjured and injured (50 dpi, freeze) TA/EDL muscle sections of control (left) and Fos cKO mice (right) (n = 5 mice per genotype per condition). Dots represent data for individual control or Fos cKO animals overlaid with mean ± SD. Student’s two-tailed unpaired (A, B, F, H, and I) and paired (J) t test and Mann-Whitney U test (E and G). Scale bars, 100 μm (A, B, and D). See also .

Article Snippet: Mouse anti-mouse PAX7 , DSHB , RRID: AB_528428.

Techniques: Control, Cell Culture, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Cell reports

Article Title: FOS licenses early events in stem cell activation driving skeletal muscle regeneration

doi: 10.1016/j.celrep.2020.108656

Figure Lengend Snippet: (A) RNA-seq (normalized read counts) showing mean (±SD) Art1 mRNA expression in fresh Fos cKO and control SCs. (B) ChIP-qPCR assays using a FOS or immunoglobulin G (IgG)-only antibody to immunoprecipitate chromatin isolated from cultured SCs ectopically expressing FOS(+FOS) or GFP (+GFP) (n = 3 independent ChIP experiments using SCs from 3 mice). 5 different probes targeted the Art1 promoter near the FOS/AP-1 DNA motif. (C) RNA-Seq (normalized read counts) showing mean (±SD) Art1 mRNA expression in fresh SCs isolated from in-situ -fixed (T0) or non-pre-fixed (standard, T3) skeletal muscle . (D) qPCR showing mean (±SD) Art1 mRNA expression (normalized to GAPDH) in fresh relative to 7-day-cultured SCs. (E) 3,000 fresh SCs were cultured for 3 days with vehicle or MIBG (50 μM). Shown are images and quantification of the mean (±SD) percentage of EdU+ nuclei among Hoechst+ SCs (top). 3,000 fresh SCs were cultured for 6 days with vehicle or MIBG (50 μM). Shown are images and quantification of the total number (mean ± SD) of Hoechst+ nuclei (bottom) (n = 4 mice each condition). (F) 3,000 fresh SCs were infected with lentivirus on the day of isolation with a non-targeting control (NTC) shRNA or one of two distinct shRNAs targeting Art1 mRNA and cultured for 6 days. Shown are images and quantification of the mean percentage (±SD) of EdU+ nuclei among Hoechst+ nuclei (top) or the total number (mean ± SD) of Hoechst+ nuclei (bottom) (n = 4 mice per condition). (G) Distribution of ADP ribosylation levels (corrected total cell fluorescent signal [CTCF]) on individual vehicle/MIBG-treated (left), control/Fos cKO (center), and shNTC/sh Art1 -expressing (right) SCs cultured for 3 days. IF was performed under non-permeabilization conditions to ensure extracellular signal. n = 250 cells (vehicle)/80 cells (MIBG) from 3 mice, n = 588 cells (control)/395 cells (Fos cKO ) from 4 mice, and n = 1,300 cells (shNTC)/695 cells ( shArt1 ) from 4 mice. Red lines represent the median value, and lower and upper black lines represent the first and third quartiles of the data. shArt1 -expressing SCs have reduced cell-surface ADP ribosylation in a small subset of the population, specifically in the first quartile (highlighted in ). Shown are three representative images of cellsurface ADP ribosylation (green) on Hoechst+ (blue) control and Fos cKO fresh SCs after 3 days in culture. (H) Experimental design. We performed two consecutive injections for 2 days (1 injection per day) of MIBG (50 μM) or vehicle into the TA muscle following a CTX injury and then harvested regenerating muscle 7 dpi. (I) Representative images showing PAX7+ SCs associated with regenerating (centrally nucleated) fibers 7 days after CTX injury. Pax7 (red), nuclei (DAPI), and Laminin (Green) are shown. (J) Quantification of the total number of PAX7+ SCs per TA/EDL muscle section in vehicle- and MIBG-treated mice (n = 5 mice per treatment). (K and L) Enumeration of the mean CSA (K) and distribution (L) of regenerating (centrally nucleated) muscle fibers in vehicle- and MIBG-treated mice at 7 dpi (n = 5 mice per condition). Two-way ANOVA with post hoc Holm-Sidak test (B), one-way ANOVA with Tukey post hoc test (F), Student’s two-tailed unpaired t test (D, E, J, and K), and Mann-Whitney U test (G and L). Scale bars represent 100 μm (E and F), 10 mm (G), and 50 μm (I). See also .

Article Snippet: Mouse anti-mouse PAX7 , DSHB , RRID: AB_528428.

Techniques: RNA Sequencing, Expressing, Control, ChIP-qPCR, Isolation, Cell Culture, In Situ, Infection, shRNA, Injection, Two Tailed Test, MANN-WHITNEY

Journal: Cell reports

Article Title: FOS licenses early events in stem cell activation driving skeletal muscle regeneration

doi: 10.1016/j.celrep.2020.108656

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse anti-mouse PAX7 , DSHB , RRID: AB_528428.

Techniques: Control, Recombinant, Lysis, Protease Inhibitor, DNA Library Preparation, cDNA Synthesis, SYBR Green Assay, Imaging, Transfection, Virus, Plasmid Preparation, Immunodetection, Magnetic Beads, Microarray, Cloning, Software

Journal: Cell reports

Article Title: IL-2 can signal via chemokine receptors to promote regulatory T cells’ suppressive function

doi: 10.1016/j.celrep.2023.112996

Figure Lengend Snippet: (A and B) Binding of soluble ICAM-1, VCAM-1, or MAdCAM-1 to CD4 T cells in the presence of PC61 (5 μg/mL) or PMA (100 nM). GFP + Tregs (A) or GFP − Tconvs (B) were analyzed. Data represent mean ± SEM (n = 7). One-way ANOVA with Bonferroni post-test. (C) Binding of soluble MAdCAM-1 to GFP + Tregs in the presence of 5 μg/mL PC61, 7D4, or 3C7 was analyzed. Data represent mean ± SEM (n = 7). One-way ANOVA with Bonferroni post-test. (D and E) The DuoLink proximity ligation assay was performed to measure the association of integrin β7 with talin1 in Tregs after PC61 stimulation. (D) Schematic illustration of the DuoLink proximity ligation assay. (E) CD4 + T cells were stimulated with PC61 or IgG in 37°C for 30 min, fixed, permeabilized, and stained with rabbit anti-β7 and mouse anti-talin, and proximity ligation assay was performed to assess the interaction between integrin β7 and talin1. GFP + Tregs and GFP − Tconvs were analyzed. Data represent mean ± SEM (n = 6). Two-tailed t test. (F) Binding of soluble MAdCAM-1 to Tregs from indicated gene-edited mice in the presence of PC61 (5 μg/mL) or PMA (100 nM). Data represent mean ± SEM (n = 7). One-way ANOVA with Bonferroni post-test. NS, not significant; **p < 0.01; ***p < 0.001.

Article Snippet: InVivoMAb rat IgG1 Isotype control, anti-trinitrophenol (Control of PC61) , BioXcell , BE0290;RRID:AB_2687813.

Techniques: Binding Assay, Proximity Ligation Assay, Staining, Two Tailed Test

Journal: Cell reports

Article Title: IL-2 can signal via chemokine receptors to promote regulatory T cells’ suppressive function

doi: 10.1016/j.celrep.2023.112996

Figure Lengend Snippet: (A and B) Tregs’ suppressive activity. (A) Tregs isolated from CD45.2 congenic Rap1a fl/fl ,Rap1b fl/fl ,Foxp3 YFP − Cre mice ( Rap1a/b TRKO ) or Foxp3 GFP mice were mixed with responder cells at the indicated Treg/responder cell ratios with either PC61 or IgG control treatment. (B) Tregs isolated from CD45.2 congenic Foxp3 GFP mice were pre-incubated with either PC61 (PC61-Pre) or 7D4 (7D4-Pre) in 37°C for 30 min, washed, and then mixed with responder cells at the indicated Treg/responder cell ratios. Responder cells are CFSE-labeled CD45.1 congenic C57BL/6 CD4 + CD25 − naive T cells activated by anti-CD3 (5 μg/mL), anti-CD28 (5 μg/mL), and IL-2. CFSE populations gated on CD45.1 + cells were analyzed by flow cytometry on day 5 to determine the division index using FlowJo software. Data represent mean ± SEM (n = 9 in A or 6 in B). Two-way ANOVA with Bonferroni post-test. (C and D) Isolated CD4 T cells were treated with PC61 or IgG for 30 min at 37°C, washed, and cultured for 5 days in complete RPMI 1640 medium with 0.6 ng/mL IL-2. Supernatants were collected and added into CFSE-labeled responder cells with anti-CD3 (5 μg/mL), anti-CD28 (5 μg/mL), and IL-2 (6 ng/mL). CFSE populations gated on CD45.1 + cells were analyzed by flow cytometry on day 5 to determine the division index using FlowJo software. Data represent mean ± SEM (n = 6). Two-way ANOVA with Bonferroni post-test. (E) The production of IL-10 and TGF-β from the supernatants of PC61 pre-treated CD4 T cells (in C and D) were determined by ELISA. Data represent mean ± SEM (n = 6). Two-way ANOVA with Bonferroni post-test. NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: InVivoMAb rat IgG1 Isotype control, anti-trinitrophenol (Control of PC61) , BioXcell , BE0290;RRID:AB_2687813.

Techniques: Activity Assay, Isolation, Control, Incubation, Labeling, Flow Cytometry, Software, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: IL-2 can signal via chemokine receptors to promote regulatory T cells’ suppressive function

doi: 10.1016/j.celrep.2023.112996

Figure Lengend Snippet: (A) Binding of soluble MAdCAM-1 to CD4 T cells treated in the presence or absence of inhibitory anti-IL-2 receptor β (TMB1) with PC61 (5 μg/mL), IL-2 (2.5 μg/mL), or PMA (100 nM). GFP + Tregs were analyzed. Data represent mean ± SEM (n = 6). One-way ANOVA with Bonferroni post-test. NS, not significant; ***p < 0.001. (B) STAT5 phosphorylation of CD4 T cells in the presence of PC61 (5 μg/mL) or IL-2 (2.5 μg/mL). GFP + Tregs were analyzed. Data are representative of at least three independent experiments. (C) STAT5 phosphorylation response of GFP + Tregs to IL-2 in the presence or absence of 100 nM baricitinib. Data represent mean ± SEM (n = 6). (D) Binding of soluble MAdCAM-1 to GFP + Tregs treated with 5 μg/mL control IgG (−) or PC61. The addition of 100 nM baricitinib did not significantly affect the response to PC61. Data represent mean ± SEM (n = 9). One-way ANOVA with Bonferroni post-test. NS, not significant. (E) Binding of soluble MAdCAM-1 to CD4 T cells treated with indicated inhibitors in the presence of PC61 (5 μg/mL) or IgG control. GFP + Tregs were analyzed. Data represent mean ± SEM (n = 6). One-way ANOVA with Bonferroni post-test. (F) Binding of soluble MAdCAM-1 to CD4 T cells treated with pertussis toxin (PTX) (100 nM) or vehicle in the presence of PC61 (5 μg/mL), CXCL12 (1 μg/mL), or PMA (100 nM). GFP + Tregs were analyzed. Data represent mean ± SEM (n = 6). One-way ANOVA with Bonferroni post-test. (G) The key signaling events that occur downstream of GPCR signaling that lead to integrin activation. (H) PTX blocks PC61 enhancement of Tregs’ suppressive activity. Tregs isolated from CD45.2 congenic Foxp3 GFP mice were mixed with responder cells at the indicated Treg/responder cell ratios in the presence of PC61 (5 μg/mL), PTX (100 nM), or PC61 together with PTX. Responder cells were CFSE-labeled CD45.1 congenic C57BL/6 CD4 + CD25 − naive T cells activated by anti-CD3 (5 μg/mL), anti-CD28 (5 μg/mL), and IL-2 (6 ng/mL). CFSE populations gated on CD45.1 + cells were analyzed by flow cytometry on day 5 to determine the division index using FlowJo software. Data represent mean ± SEM (n = 4). Two-way ANOVA with Bonferroni post-test. NS, not significant; **p < 0.01; ***p < 0.001.

Article Snippet: InVivoMAb rat IgG1 Isotype control, anti-trinitrophenol (Control of PC61) , BioXcell , BE0290;RRID:AB_2687813.

Techniques: Binding Assay, Phospho-proteomics, Control, Activation Assay, Activity Assay, Isolation, Labeling, Flow Cytometry, Software

Journal: Cell reports

Article Title: IL-2 can signal via chemokine receptors to promote regulatory T cells’ suppressive function

doi: 10.1016/j.celrep.2023.112996

Figure Lengend Snippet: (A) The RNA expression level of chemokine receptors in murine splenic Tregs ( http://rstats.immgen.org/Skyline/skyline.html ). (B) Jurkat cells stably expressing mouse CD25 (mCD25-Jurkat) were transfected with CCR7 or CXCR4 siRNA or scrambled-siRNA (2 μM) by Amaxa Nucleofector. 72 h post-transfection, the cells were stimulated with PC61 (5 μg/mL), CXCL12 (1 μg/mL), or CCL21 (1 μg/mL), and binding to soluble VCAM-1 was measured. (C and D) Co-immunoprecipitation of CCR7 with CD25 after PC61 stimulation. (C) Experimental scheme. (D) Jurkat cells stably expressing mouse CD25 (mCD25-Jurkat) were treated with IgG control, PC61 (5 μg/mL), 3C7 (5 μg/mL), or PC61 combined with PTX (100 nM) at 37°C for 30 min and lysed. CD25 was isolated by immunoprecipitation (IP). CCR7 was analyzed by western blotting. Data are representative of at least three independent experiments. The PTX-treated cells were analyzed on the same SDS-PAGE as other samples; however, irrelevant intervening lanes were excised. (E and F) The DuoLink proximity ligation assay was performed to measure the association of CCR7 with CD25 in primary untransfected Tregs after PC61 stimulation. (E) Schematic illustration of the DuoLink proximity ligation assay. (F) CD4 T cells were stimulated with murinized PC61 (mPC61) or mIgG at 37°C for 30 min in the presence or absence of PTX and then were fixed, permeabilized, and stained with rabbit anti-CCR7, and a proximity ligation assay was performed to assess the interaction between CCR7 and CD25. GFP + Tregs (left) and GFP − Tconvs (right) were analyzed. Data represent mean ± SEM (n = 9). One-way ANOVA with Bonferroni post-test. NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: InVivoMAb rat IgG1 Isotype control, anti-trinitrophenol (Control of PC61) , BioXcell , BE0290;RRID:AB_2687813.

Techniques: RNA Expression, Targeted Proteomics, Stable Transfection, Expressing, Transfection, Binding Assay, Immunoprecipitation, Control, Isolation, Western Blot, SDS Page, Proximity Ligation Assay, Staining

Journal: Cell reports

Article Title: IL-2 can signal via chemokine receptors to promote regulatory T cells’ suppressive function

doi: 10.1016/j.celrep.2023.112996

Figure Lengend Snippet: (A) Binding of soluble MAdCAM-1 to thrice-washed (Wash) or unwashed (No wash) CD4 + T cells following addition of PC61 (5 μg/mL) in the presence or absence of 1.25 μg/mL IL-2. Data for GFP + Tregs are depicted and represent mean ± SEM (n = 5). One-way ANOVA with Bonferroni post-test. (B) CD4 + T cells were pre-treated with 5 μg/mL 7D4, 3C7, or Ig controls before addition of PC61 (5 μg/mL). Binding of soluble MAdCAM-1 to GFP + Tregs is depicted. Data represent mean ± SEM (n = 7). One-way ANOVA with Bonferroni post-test. (C) CD4 + T cells were stimulated with PC61 (5 μg/mL) or IgG control in the presence or absence of an antibody that blocks IL-2 binding to CD25 (IL-2 Neut) (2 μg/mL), and MAdCAM-1 binding to GFP + Tregs was measured. Data represent mean ± SEM (n = 7). One-way ANOVA with Bonferroni post-test. (D) Structural model of the association of IL-2 and murine CD25. R57 and L63 are predicted to mediate interactions between IL-2 and CD25. (E) IL-2 binding to CD25 is required for PC61-induced association of Cd25 with CCR7. 293T cells stably expressing hemagglutinin (HA)-tagged mouse CD25 or CD25(R57.L63E) (CD25(2E)) were treated with PC61 (5 μg/mL), PC61 plus IL-2 (5 μg/mL), 3C7 plus IL-2, or IL-2 alone at 37°C for 30 min and lysed. HA-CD25 and HA-CD25(2E) were captured by IP with anti-HA antibody. CCR7 was detected by immunoblotting. Data are representative of at least three independent experiments. (F) CCR7 is required for the association of IL-2 with PC61-bound CD25. 293T cells stably expressing HA-tagged mouse CD25 were transfected with siRNA against CCR7 or control siRNA and after 48 h were incubated at 37°C for 30 min in the presence of IL-2 (2.5 μg/mL) and PC61 (5 μg/mL) or control IgG. Following lysis, HA-CD25 was captured by IP, and the presence of IL-2, CCR7, and CD25 was ascertained by immunoblotting. Data are representative of three independent experiments.

Article Snippet: InVivoMAb rat IgG1 Isotype control, anti-trinitrophenol (Control of PC61) , BioXcell , BE0290;RRID:AB_2687813.

Techniques: Binding Assay, Control, Stable Transfection, Expressing, Western Blot, Transfection, Incubation, Lysis

Journal: Cell reports

Article Title: IL-2 can signal via chemokine receptors to promote regulatory T cells’ suppressive function

doi: 10.1016/j.celrep.2023.112996

Figure Lengend Snippet:

Article Snippet: InVivoMAb rat IgG1 Isotype control, anti-trinitrophenol (Control of PC61) , BioXcell , BE0290;RRID:AB_2687813.

Techniques: Control, Recombinant, Purification, Produced, Virus, Bacteria, Electron Microscopy, Emulsion, Staining, Cell Culture, Expressing, Transfection, Cell Isolation, Enzyme-linked Immunosorbent Assay, Endotoxin Assay, cDNA Synthesis, Mutagenesis, Plasmid Preparation, Software

Journal: eLife

Article Title: Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery

doi: 10.7554/eLife.31101

Figure Lengend Snippet: ( A ) Representative western blots of endocytic or adhesion molecules. Cell lysates obtained from cortical neurons grown on substrates with differing stiffness were subjected to immunoblotting with antibodies to indicated proteins. ( B ) Summary histograms, all from experiments similar to that described in A , showing greater (>2 fold) abundance of paxillin, phospho-paxillin Y118 (p-paxillin), myosin VI (myo6), CIP4 and clathrin heavy chain (CHC) proteins in neurons grown on soft substrates (0.1 and 1 kPa) compared to those grown on stiff substrates (20 kPa and glass). Note the inverse expression of endocytic factors and adhesion molecules (i.e., talin1/2, integrinβ1, vinculin and p-FAK Y397 ). Data represent mean ±SEM (n ≥ 3, normalized to control actin; compared to 0.1 kPa cultures; *p<0.05; **p<0.01; ***p<0.001; t -test). ( C–F ) Paxillin co-localizes with the endocytosis complex at neuronal growth cones on soft substrates. ( C ) Representative confocal images of 16 hr neurons on 0.1 kPa gels co-immunostained with antibodies against paxillin (Green in merge panel), adaptor-associated kinase1 (AAK1, Red in merge panel), p-FAK Y397 (Red in merge panel), or F-actin, as indicated. Right panels show the region of interest ROI (marked by numbers) of neurite tips represented at higher magnification. Bar: 20 μm. ( D and E ) Similar to C , except the resolution of images has been enhanced (~1.7X higher) using Airyscan. Note that surface rendering was applied at neurite tips to more clearly show co-localization (yellow) of paxillin with indicated factors. Bar: 20 μm. ( F ) Histograms, all from experiments similar to those described in d and e , summarizing percentages of paxillin co-localized with indicated endocytic or adhesion factors on hydrogels. Data represent percentages (±SEM, n = 15 neurons for each set of experiments; ROI, 5 × 5 µm within one lamellipodium; **p<0.01; ***p<0.001; ****p<0.0001; t test).

Article Snippet: For paxillin shRNA constructs, 29-oligonucleotide duplexes targeting rat paxillin cDNA sequences (PXN-shRNA A targeting position 1505, PXN-shRNA B targeting position 1571, PXN-shRNA C targeting position 346, and PXN-shRNA D targeting position 952 of the corresponding paxillin sequence) and a control non-effective scrambled shRNA cassette (5’- CACAAGCTGGAGTACAACTACAACAGCCA-3’) were cloned into pGFP-C-shLenti vector (OriGene Technologies, Rockville, MD).

Techniques: Western Blot, Expressing

Journal: eLife

Article Title: Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery

doi: 10.7554/eLife.31101

Figure Lengend Snippet: ( A–C ) Membrane flotation assay of embryonic brain ( A ) and heart ( B ) tissue lysates. Representative western blot of gradient fractionations probed with antibodies against endocytic factors (Rab5 and CHC), adhesion-associated molecules (vinculin, and phospho-FAK Y397 ), paxillin, and actin, as indicated. Histogram in A reflects quantitative measurements of the protein levels (±SEM; n = 3 independent experiments; **p<0.01; compared to that of paxillin; multiple t test for each fraction) found in the floating fraction (collective measurement from fraction 10 to fraction 18) out of the total gradient fractions. ( C ) Traces depict the differential protein levels (±SEM; from all experiments similar to that described in A and B, ***p<0.001; multiple t test) in each gradient fraction between embryonic brain and heart lysates, as indicated.

Article Snippet: For paxillin shRNA constructs, 29-oligonucleotide duplexes targeting rat paxillin cDNA sequences (PXN-shRNA A targeting position 1505, PXN-shRNA B targeting position 1571, PXN-shRNA C targeting position 346, and PXN-shRNA D targeting position 952 of the corresponding paxillin sequence) and a control non-effective scrambled shRNA cassette (5’- CACAAGCTGGAGTACAACTACAACAGCCA-3’) were cloned into pGFP-C-shLenti vector (OriGene Technologies, Rockville, MD).

Techniques: Western Blot

Journal: eLife

Article Title: Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery

doi: 10.7554/eLife.31101

Figure Lengend Snippet: Representative image ( A ) and kymograph ( B ) of paxillin-mCherry trajectories (x-axis, distance; y-axis, time) generated from 180 s time-lapse images (90 frames; 0.5 frames per second), showing bidirectional movement of paxillin-mCherry along the axon (dashed line). Summary of directionality ( C ) and velocity distribution ( D ) of paxillin-mCherry along axons from all experiments similar to that described in B. Data represent means ±SEM (n = 15 cells).

Article Snippet: For paxillin shRNA constructs, 29-oligonucleotide duplexes targeting rat paxillin cDNA sequences (PXN-shRNA A targeting position 1505, PXN-shRNA B targeting position 1571, PXN-shRNA C targeting position 346, and PXN-shRNA D targeting position 952 of the corresponding paxillin sequence) and a control non-effective scrambled shRNA cassette (5’- CACAAGCTGGAGTACAACTACAACAGCCA-3’) were cloned into pGFP-C-shLenti vector (OriGene Technologies, Rockville, MD).

Techniques: Generated

Journal: eLife

Article Title: Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery

doi: 10.7554/eLife.31101

Figure Lengend Snippet: ( A ) Paxillin preferentially binds endocytic factors in neurons grown on soft substrates. Paxillin-associated complexes were immunoprecipitated (IP) in lysates made from E17.5 rat brain or from cortical neuronal cultures grown on different substrates using a specific paxillin antibody and were then detected by western blot analysis. Normal rabbit IgG (‘IgG’) served as a negative control. Histograms show the opposing binding preference of paxillin toward CIP4/CHC or vinculin when grown on soft (0.1 kPa or 1 kPa) versus rigid (20 kPa or glass) substrates. Data represent mean intensity ±SEM (n = 3 independent experiments; *p<0.05; t- test). ( B ) Western blot showing direct interaction of paxillin with CIP4. Bacterially expressed His-CIP4 was purified by fast protein liquid chromatography and subjected to a GST pull-down assay using GST-PXN FL , GST-PXN ∆LIM3-4 or GST alone. Precipitants were analyzed by western blotting with antibodies specific to CIP4. Histograms summarize protein levels as determined by immunoblotting of full-length (His-CIP4) or F-BAR domain-deleted (His-ΔF-BAR) CIP4 pulled-down by GST-paxillin variants (±SEM, n = 3; normalized to the corresponding GST-PXN FL or GST-PXN ∆LIM3-4 inputs; **p<0.01, t test). ( C and D ). Mapping of paxillin domains interacting with CIP4 or vinculin. ( C ) GST pull-down and immunoblotting of vinculin, myc-CIP4, and CHC in lysates of myc-CIP4-expressing HEK293T cells. Histograms reflect quantification of levels of proteins pulled-down by GST fusions of full-length (“FL“) or LIM domain- and/or LD motif-deleted forms of paxillin, all from experiments similar to those shown in top panels (±SEM, n ≥ 3 independent experiments; *p<0.05; **p<0.01; ***p<0.001; t test). ( D ) Schematic of GST fusion proteins used in c . Table summarizing relative CIP4 or vinculin (‘Vin’) binding by paxillin deletion mutants or full-length protein. Solid lines mark primary sites of interaction, and dashed lines mark accessory interaction motifs for strong binding to vinculin or CIP4. Binding strength relative to full-length paxillin indicated as: ‘++++' >75% > ‘+++' >50% > ‘++' >25% > '+' >5% > '+/-'. ( E ) In vitro protein interaction and competitive binding assays in HEK293T cells transfected with various amounts (1, 6, and 12 µg) of plasmids encoding myc-tagged CIP4 protein (myc-CIP4) and/or control vectors, as indicated. Cell lysates were subjected to a GST pull-down assay with GST-PXN FL or GST alone, and immunoblotted with vinculin and myc antibodies. Line chart depicts averaged protein levels as determined by immunoblotting of CIP4 or vinculin pulled-down by GST-PXN FL (±SEM, n = 4; normalized to band intensity of corresponding GST-paxillin variant). ( F ) In vivo protein interaction and competitive binding assays in HEK293T cells transfected with various amounts (7.5 μg and 15 μg) of plasmids encoding the F-BAR domain (‘F-BAR’) alone or F-BAR-domain-deleted (‘ΔN’-F-BAR’) CIP4 and/or control vectors, as indicated. Cell lysates were immunoprecipitated by paxillin antibodies and blotted with myc or vinculin antibodies. Histograms show relative protein levels as determined by immunoblotting of vinculin co-immunoprecipitated by paxillin antibodies (±SEM, n = 3; *p<0.05, t test).

Article Snippet: For paxillin shRNA constructs, 29-oligonucleotide duplexes targeting rat paxillin cDNA sequences (PXN-shRNA A targeting position 1505, PXN-shRNA B targeting position 1571, PXN-shRNA C targeting position 346, and PXN-shRNA D targeting position 952 of the corresponding paxillin sequence) and a control non-effective scrambled shRNA cassette (5’- CACAAGCTGGAGTACAACTACAACAGCCA-3’) were cloned into pGFP-C-shLenti vector (OriGene Technologies, Rockville, MD).

Techniques: Immunoprecipitation, Western Blot, Negative Control, Binding Assay, Purification, Fast Protein Liquid Chromatography, Pull Down Assay, Expressing, In Vitro, Transfection, Variant Assay, In Vivo

Journal: eLife

Article Title: Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery

doi: 10.7554/eLife.31101

Figure Lengend Snippet: Paxillin-associating protein identified by liquid chromatography-tandem mass spectrometry.

Article Snippet: For paxillin shRNA constructs, 29-oligonucleotide duplexes targeting rat paxillin cDNA sequences (PXN-shRNA A targeting position 1505, PXN-shRNA B targeting position 1571, PXN-shRNA C targeting position 346, and PXN-shRNA D targeting position 952 of the corresponding paxillin sequence) and a control non-effective scrambled shRNA cassette (5’- CACAAGCTGGAGTACAACTACAACAGCCA-3’) were cloned into pGFP-C-shLenti vector (OriGene Technologies, Rockville, MD).

Techniques: Chromatography, Transduction

Journal: eLife

Article Title: Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery

doi: 10.7554/eLife.31101

Figure Lengend Snippet: ( A ) Structures of the vinculin tail (PDB ID: 1QKR; top panel) and the CIP4 F-BAR domain (PDB ID: 2EFK; bottom panel) constructed by JMOL, version 14.4.4. Yellow marks indicate the predicted paxillin-binding subdomain (PBS) for vinculin (951K to 970Q) or CIP4 (35R to 55P). Secondary structures were predicted by the DSSP or STRIDE databases. ( B ) Mapping of paxillin domains interacting with CIP4 or vinculin. GST pull-down and immunoblotting of vinculin and myc-CIP4 in lysates of myc-CIP4-expressing HEK293T cells. Histograms reflect quantification of levels of proteins pulled-down by GST fusions of full-length ('FL') or LD motif-deleted forms of paxillin, all from experiments similar to those shown in top panels (±SEM, n ≥ 3 independent experiments; **p<0.01; ***p<0.001; multiple t test). Schematic of GST fusion proteins and table summarizing relative CIP4 or vinculin (‘Vin’) binding by paxillin deletion mutants or full-length protein shown in the top panel. Binding strength relative to full-length paxillin indicated as: ‘++++' >75% > ‘+++' >50% > ‘++' >25% > '+' >5% > '+/-'. Note that although CIP4 primarily associates with LIM domains, deletion of the paxillin LD1-3 domain reduced its affinity for CIP4. ( C ) Mapping of CIP4 domains interacting with paxillin. GST fusion proteins of full-length CIP4 (‘FL’) or its variants with F-BAR domain, HR1 and/or SH3 domain truncations were subjected to GST pull-down assays in HEK293T cell lysates, followed by immunoblotting for paxillin and actin. Histogram reflects quantitative measurement of relative protein levels (±SEM, n ≥ 3 independent experiments; compared to that of FL experiment; **p<0.01; t -test) pulled down by GST-CIP4, as indicated. ( D ) Schematic of GST fusion proteins used in B . Table summarizes relative paxillin binding by CIP4 deletion mutants or full-length protein. Binding strength relative to that of full-length CIP4 is represented as: ‘++++' >75% > ‘+++' >50% > ‘++' >25% > '+'. ( E ) In vivo protein interactions in HEK293T cells co-transfected with plasmids encoding paxillin-GFP and myc-tagged CIP4 deletion mutants or full-length protein, as indicated. Cell lysates were immunoprecipitated by myc antibody and blotted with paxillin or myc antibodies. Histograms show relative protein levels as determined by immunoblotting for paxillin co-immunoprecipitated by myc antibody. Data represents mean (±SEM from more than three independent experiments; compared to that of F-BAR experiment; *p<0.05; t -test). ( F ) In vitro protein interaction and competitive binding assays in HEK293T cells transfected with various amounts (1, 6, and 12 µg) of plasmids encoding myc-tagged CIP4 protein (myc-CIP4) and/or control vectors, as indicated. Cell lysates were subjected to a GST pull-down assay with GST-PXN FL , GST-PXN ∆LD1 , GST-PXN ∆LIM3-4 or GST alone, and immunoblotted with vinculin and myc antibodies. Line chart depicts averaged protein levels as determined by immunoblotting of CIP4 or vinculin pulled-down by GST-PXN variant (±SEM, n = 3–4; normalized to band intensity of corresponding GST-paxillin variant).

Article Snippet: For paxillin shRNA constructs, 29-oligonucleotide duplexes targeting rat paxillin cDNA sequences (PXN-shRNA A targeting position 1505, PXN-shRNA B targeting position 1571, PXN-shRNA C targeting position 346, and PXN-shRNA D targeting position 952 of the corresponding paxillin sequence) and a control non-effective scrambled shRNA cassette (5’- CACAAGCTGGAGTACAACTACAACAGCCA-3’) were cloned into pGFP-C-shLenti vector (OriGene Technologies, Rockville, MD).

Techniques: Construct, Binding Assay, Western Blot, Expressing, Protein Binding, In Vivo, Transfection, Immunoprecipitation, In Vitro, Pull Down Assay, Variant Assay

Journal: eLife

Article Title: Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery

doi: 10.7554/eLife.31101

Figure Lengend Snippet: ( A ) Western blot showing efficiency of shRNA-mediated paxillin knockdown. Mouse neuroblastoma Neuro-2a (N2a) cells were transfected with plasmids encoding scrambled shRNA (‘scr’) or one of four paxillin shRNAs (sequence A, B, C, or D) targeting to different regions of the paxillin sequence. Summary histograms showing that shRNA D exerted a ~2-fold suppression on paxillin expression and it had no effect on vinculin expression. Data represent mean ±SEM (n = 5, normalized to control actin, compared to that of scr experiment; *p<0.05; t -test **p<0.01, ****p<0.0001; t test). ( B ) Images of neurons transfected with plasmids encoding scrambled shRNA or paxillin shRNAs (shRNA C + shRNA D), immunostained with antibody specific against paxillin. Right-most panels show the region of interest (ROI, dashed box) of neurite tips at a higher magnification, with the intensity of paxillin staining coded by pseudocolors in a linear scale. Bar: 5 μm. Dot plot reflects quantification of paxillin immunostaining intensity (±SEM, n = 20 cells each, normalized to EGFP intensity and relative to scramble control; ****p<0.0001; one-way ANOVA with Dunnett’s post hoc test).

Article Snippet: For paxillin shRNA constructs, 29-oligonucleotide duplexes targeting rat paxillin cDNA sequences (PXN-shRNA A targeting position 1505, PXN-shRNA B targeting position 1571, PXN-shRNA C targeting position 346, and PXN-shRNA D targeting position 952 of the corresponding paxillin sequence) and a control non-effective scrambled shRNA cassette (5’- CACAAGCTGGAGTACAACTACAACAGCCA-3’) were cloned into pGFP-C-shLenti vector (OriGene Technologies, Rockville, MD).

Techniques: Western Blot, shRNA, Transfection, Sequencing, Expressing, Staining, Immunostaining

Journal: eLife

Article Title: Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery

doi: 10.7554/eLife.31101

Figure Lengend Snippet: ( A ) Paxillin knockdown suppresses the endocytic activity of neurons grown on 0.1 kPa gels. Representative time-lapse images of FM4-64 uptake in 3-DIV neurons on substrates of varying elasticity. Hippocampal neurons on 0.1 kPa gels were transduced with lentiviral particles harboring an shRNA-resistant construct (‘PXN-R’) and/or constructs harboring scrambled control or paxillin (‘PXN shRNA C + D’) shRNA at 5 hr after cell plating. Dashed circles surround the region of interest (ROI) in quantitative FM4-64 measurements. Bar: 20 μm. ( B ) Similar to A , except constructs encoding GFP fusions of wild-type paxillin (‘PXN WT -GFP’; B1 and B2 ) or the corresponding LIM domain deletion mutant (‘PXN LD1-3 -GFP’; B3 ) were used for lentiviral transduction. Asterisk: non-transduced neighboring cells. Arrows: neurons expressing GFP-tagged paxillin proteins. Bar: 20 μm. ( C–E ) Quantitative measurements of cumulative FM4-64 intensity (±SEM, n > 3 independent experiments, 7–12 cells for each set of experiments; *p < 0.05; **p<0.001; ***p<0.0001; compared to control groups; multiple t tests), all from experiments similar to those described in A and B . Note that ectopic expression of wild-type paxillin, but not PXN LD1-3 , restored rapid endocytic FM4-64 uptake on 20 kPa stiff gels.

Article Snippet: For paxillin shRNA constructs, 29-oligonucleotide duplexes targeting rat paxillin cDNA sequences (PXN-shRNA A targeting position 1505, PXN-shRNA B targeting position 1571, PXN-shRNA C targeting position 346, and PXN-shRNA D targeting position 952 of the corresponding paxillin sequence) and a control non-effective scrambled shRNA cassette (5’- CACAAGCTGGAGTACAACTACAACAGCCA-3’) were cloned into pGFP-C-shLenti vector (OriGene Technologies, Rockville, MD).

Techniques: Activity Assay, Transduction, shRNA, Construct, Mutagenesis, Expressing

Journal: eLife

Article Title: Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery

doi: 10.7554/eLife.31101

Figure Lengend Snippet: ( A ) QD-BDNF internalization assay on HEK293T cells co-transfected with expression vectors encoding the BDNF receptor TrkB (Red), together with shRNA constructs (Green) and/or wild-type paxillin, as indicated. Histograms showing that co-transfection of paxillin shRNA C and shRNA D significantly reduced QD-BDNF internalization (as reflected by an ~8-fold reduction in the quantity of intracellular QD-BDNF). Data represent means ±SEM (n ≥ 3 independent experiments, 26–52 cells for each set of experiments; compared to scrambled shRNA control; ***p<0.001; ANOVA with Dunnett’s post hoc test). ( B ) Similar to A , except cells were pre-incubated with or without the dynamin inhibitor dynasore (50 μM, 30 min). Histograms show that dynamin is required for paxillin to promote QD-BDNF internalization. Data represent means ±SEM (n ≥ 3 independent experiments, 21–58 cells for each set of experiments; compared to GFP control; ***p<0.001; ANOVA with Dunnett’s post hoc test).

Article Snippet: For paxillin shRNA constructs, 29-oligonucleotide duplexes targeting rat paxillin cDNA sequences (PXN-shRNA A targeting position 1505, PXN-shRNA B targeting position 1571, PXN-shRNA C targeting position 346, and PXN-shRNA D targeting position 952 of the corresponding paxillin sequence) and a control non-effective scrambled shRNA cassette (5’- CACAAGCTGGAGTACAACTACAACAGCCA-3’) were cloned into pGFP-C-shLenti vector (OriGene Technologies, Rockville, MD).

Techniques: Transfection, Expressing, shRNA, Construct, Cotransfection, Incubation

Journal: eLife

Article Title: Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery

doi: 10.7554/eLife.31101

Figure Lengend Snippet: ( A and B ). Paxillin knockdown impairs lamellipodium segmentation and neurite formation in neuronal cultures on 0.1 kPa gels. ( A ) Images of 16 hr hippocampal neurons transfected with scrambled control or paxillin siRNA at 30 min after cell plating on 0.1 kPa gels, followed by phalloidin staining for F-actin (Red) and immunostaining for the neuronal marker Tuj-1 (Green) at 16 hr after cell plating. ( B ) Histograms showing that paxillin knockdown significantly decreased the percentage of neurite-bearing neurons (left y-axis) and increased the percentage of neurons exhibiting the BL phenotype (right y-axis) in 0.1 kPa cultures. Data represent mean (±SEM; n > 3 independent experiments, >250 cells for each group; *p<0.05; **p<0.01; t test). ( C ) Similar to A , except that 16 hr neuronal cultures were immunostained with antibodies against active Rac1-GTP (Gray). Fluorescence intensity of active Rac1 (boxed region) is coded by pseudocolors in the linear scale (right panel in C ). Scale bar: 20 μm. ( D ) Quantification of active Rac1 levels at segmented lamellipodia (averaged pixel value of a 2 µm X 2 µm area) in 0.1 kPa cultures transfected with paxillin-siRNA or control scrambled siRNA. Data represent mean ±SEM (n > 3 independent experiments, >250 cells for each group; ***p<0.001; ANOVA with Dunnett’s post hoc test). ( E ) Paxillin knockdown promotes aberrant neurite formation and cortical neuron migration in vivo. Fluorescence images of E20 rat cortices transfected in utero at E17.5 with IRES constructs harboring EGFP plus scrambled-shRNA control, paxillin shRNA C + D, and/or shRNA C + D resistant PXN-R. The middle panels show 4x magnifications of boxed regions of the corresponding E20 cortex in the top panels. Bottom panels show sample tracings of 2D projections from confocal images of eight typical cortical neurons in the subventricular zone (‘SVZ’) of the corresponding E20 cortex. Bar, 100 μm. ( F ) Histograms showing the percentage (±SEM, n = 3 cortices each; *p<0.05, two-tailed t test) of neurons residing in the cortical plate (‘CP’), intermediate zone (‘IZ’), or subventricular zone (‘SVZ’) regions. ( G ) Calculation of the percentages (±SEM, n > 150 cells per cortex,>5 cortices each; ***p<0.001, multiple t test) of transfected cortical neurons exhibiting unipolar/bipolar processes or no process (‘none’; arrowheads in e ) in the cortical SVZ/IZ region for cortices described in e . Datasets (connected by dashed lines) showing significant differences are marked. ( H ) Schematic illustrating the proposed substrate elasticity-controlled, paxillin-dependent bistable mechanism, comprising a genetic response and mutual inhibition of endocytosis by adhesion and vice versa. Growth on a soft substrate shifts neurons to a Rac1-activated neuritogenic state.

Article Snippet: For paxillin shRNA constructs, 29-oligonucleotide duplexes targeting rat paxillin cDNA sequences (PXN-shRNA A targeting position 1505, PXN-shRNA B targeting position 1571, PXN-shRNA C targeting position 346, and PXN-shRNA D targeting position 952 of the corresponding paxillin sequence) and a control non-effective scrambled shRNA cassette (5’- CACAAGCTGGAGTACAACTACAACAGCCA-3’) were cloned into pGFP-C-shLenti vector (OriGene Technologies, Rockville, MD).

Techniques: Transfection, Staining, Immunostaining, Marker, Fluorescence, Migration, In Vivo, In Utero, Construct, shRNA, Two Tailed Test, Inhibition

Journal: eLife

Article Title: Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery

doi: 10.7554/eLife.31101

Figure Lengend Snippet: ( A ) Representative fluorescent images of transfected IUE neurons (yellow arrows) expressing control EGFP and/or a paxillin deletion mutation (Green), cultured on 0.1 kPa ( A1 ) or 20 kPa ( A2 ) gels for 16 hr, and stained with phalloidin (Red), DAPI (Blue), and antibodies against Tuj-1 (Gray). ( B ) Histograms summarize the percentages (±SEM; *p<0.05; **p<0.001, one-way ANOVA with Dunnett’s post hoc test) of 16 hr neurons bearing segmented lamellipodia (‘SL’) or broad lamellipodia ('BL’) in neurons expressing different paxillin deletion mutations, as indicated. The top panel shows schematic of the neurite phenotype switch observed in the PXN∆LIM3-4 expressing neuron on 0.1 kPa gel and the PXN∆LD1 expressing neuron on 20 kPa gel.

Article Snippet: For paxillin shRNA constructs, 29-oligonucleotide duplexes targeting rat paxillin cDNA sequences (PXN-shRNA A targeting position 1505, PXN-shRNA B targeting position 1571, PXN-shRNA C targeting position 346, and PXN-shRNA D targeting position 952 of the corresponding paxillin sequence) and a control non-effective scrambled shRNA cassette (5’- CACAAGCTGGAGTACAACTACAACAGCCA-3’) were cloned into pGFP-C-shLenti vector (OriGene Technologies, Rockville, MD).

Techniques: Transfection, Expressing, Mutagenesis, Cell Culture, Staining

Journal: eLife

Article Title: Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery

doi: 10.7554/eLife.31101

Figure Lengend Snippet: Representative Western blots ( A ) and quantitative analysis ( B ) of embryonic E18 brain, heart and liver lysates. Note that endocytic factors (CHC, Rab5 and CIP4) are more abundant in brain than in heart. Data represent mean ±SEM in more than three independent experiments. ( C ) Pie charts depicting the relative levels of adhesion and endocytic factors in brain, cardiac and hepatic tissues at E18, from all experiments similar to that described in B. The size of each pie slice is proportional to the sum of adhesion molecules (including integrin β1, talin1/2, vinculin and paxillin) or endocytic accessory factors (including paxillin, CIP4, CHC and Rab5) over all proteins measured. Paxillin and actin are assigned to dual function (endocytosis and adhesion). Note that endocytic factors are highly abundant in embryonic brain tissue.

Article Snippet: For paxillin shRNA constructs, 29-oligonucleotide duplexes targeting rat paxillin cDNA sequences (PXN-shRNA A targeting position 1505, PXN-shRNA B targeting position 1571, PXN-shRNA C targeting position 346, and PXN-shRNA D targeting position 952 of the corresponding paxillin sequence) and a control non-effective scrambled shRNA cassette (5’- CACAAGCTGGAGTACAACTACAACAGCCA-3’) were cloned into pGFP-C-shLenti vector (OriGene Technologies, Rockville, MD).

Techniques: Western Blot

Journal: eLife

Article Title: Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery

doi: 10.7554/eLife.31101

Figure Lengend Snippet:

Article Snippet: For paxillin shRNA constructs, 29-oligonucleotide duplexes targeting rat paxillin cDNA sequences (PXN-shRNA A targeting position 1505, PXN-shRNA B targeting position 1571, PXN-shRNA C targeting position 346, and PXN-shRNA D targeting position 952 of the corresponding paxillin sequence) and a control non-effective scrambled shRNA cassette (5’- CACAAGCTGGAGTACAACTACAACAGCCA-3’) were cloned into pGFP-C-shLenti vector (OriGene Technologies, Rockville, MD).

Techniques: Recombinant

Journal: iScience

Article Title: miRNA-194-3p represses NF-κB in gliomas to attenuate iPSC genes and proneural to mesenchymal transition

doi: 10.1016/j.isci.2023.108650

Figure Lengend Snippet: miR-194-3p, TAB2 and iPSC gene expression in GSC vs. ML counterparts, miR-194-3p modulates NF-κB activation in GSC vs. ML matched pairs, TAB2 expression correlates with P and M subtype (A) The expression levels of miR-194-3p in matched pair ML and GSC cell lines. Data are shown as mean ± SEM. Statistical significance was tested using two-tailed unpaired t-test. (B) Relative expression levels of TAB2 and iPSC markers in matched GSC and ML pairs. Data are shown as mean ± SEM. Statistical significance was tested using two-tailed unpaired t-test. (C) Top: Quantification of TAB2 protein levels in panel of ML and GSC cell lines. Data are shown as mean ± SEM. Statistical significance was tested using two-tailed unpaired t-test. Middle: TAB2 protein levels in OSU13, OSU20 and OSU53 matched pair ML and GSC cell lines. Bottom: TAB2 protein levels in commercially available GBM cell lines; Corresponding mRNA expression are represented above images. (D) Western blot analysis of NF-κB activation in matched pair ML and GSC cell lines. (E) NF-κB activation following the transfection of 100 nM miR-194-3p mimic and inhibitor in U87 ML and OSU68 ML cells. (F) The heatmap represents the relative expression of TAB2, mesenchymal and proneural markers in the GSC cell lines. Data is row-normalized data. (G) Expression levels of proneural and mesenchymal markers in miR-194-3p isogenic cells. Data are shown as mean ± SEM. Statistical significance was tested using two-tailed unpaired t-test. ns not significant; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: Taqman gene expression assay probes were used to measure the mRNA levels of 194-1 (Thermo Fisher: Cat# Hs04231530_s1), 194-2 (Thermo Fisher: Cat# Hs04331541_s1), ALDH1A3 (Thermo Fisher: Cat# Hs00167476_m1), CD44 (Thermo Fisher: Cat# Hs05662929_s1), LYN (Thermo Fisher: Cat# Hs01015818_g1), WT1 (Thermo Fisher: Cat# Hs01103751_m1), CD133 (Thermo Fisher: Cat# Hs01009259_m1), Nestin (Thermo Fisher: Cat# Hs04187831_g1), OLIG1 (Thermo Fisher: Cat# Hs00907227_s1), SOX2 (Thermo Fisher: Cat# Hs04234836_s1), klf4 (Thermo Fisher: Cat# Hs00358836_m1), OCT4 (Thermo Fisher: Cat# Hs00999632_g1), FUT4 (Thermo Fisher: Cat# Hs01106466_s1), NCAM1 (Thermo Fisher: Cat# Hs00941830_m1), Nanog (Thermo Fisher: Cat# Hs02387400_g1), TAB2 (Thermo Fisher: Cat# Hs00248373_m1), CMYC (Thermo Fisher: Cat# Hs00153408_m1), DOCK5 (Thermo Fisher: Cat# Hs07287975_m1), FAS (Thermo Fisher: Cat# Hs00236330_m1), HFE (Thermo Fisher: Cat# Hs00373474_m1), LGALS3 (Thermo Fisher: Cat# Hs07288627_m1), MAL (Thermo Fisher: Cat# Hs00707014_s1), RHOF (Thermo Fisher: Cat# Hs00368032_m1), SOWAHA (Thermo Fisher: Cat# Hs00703106_s1), C21orf91 (Thermo Fisher: Cat# Hs05040994_s1), TRIM23 (Thermo Fisher: Cat# Hs01106626_m1).

Techniques: Gene Expression, Activation Assay, Expressing, Two Tailed Test, Western Blot, Transfection

Journal: iScience

Article Title: miRNA-194-3p represses NF-κB in gliomas to attenuate iPSC genes and proneural to mesenchymal transition

doi: 10.1016/j.isci.2023.108650

Figure Lengend Snippet: Luciferase reporter assay confirming inhibition of TAB2 by miR-194-3p, expression level of TAB2, NF-κB, and iPSC markers in U87-TAB2 isogenic cells (A) Schematic of the TAB2 3′ UTR miRNA reporter system. Sequences representing the TAB2 3′ UTR and a mismatched control were cloned into the reporter system downstream of the firefly luciferase protein (PGK). The Renilla luciferase protein was driven off a second promoter (SV40) and used as a control for transfection efficiency. (B) The miRNA reporter vectors were transfected in U87 cells for 24 h before treating with 100 nM miR-194-3p mimic or inhibitor. Activity is represented by the amount of firefly luciferase normalized to the amount of Renilla luciferase. Data are shown as mean ± SEM. Statistical significance was tested using two-way ANOVA (DF = 16). (C) U87-EV and U87-194-3p-OE were transfected using the same conditions as B. Data are shown as mean ± SEM. Statistical significance was tested using two-way ANOVA (DF = 12). (D) Relative mRNA expression of TAB2 in U87 cells transduced with shTAB2. (E) Relative TAB2, NF-κB, and iPSC marker expression levels in TAB2 isogenic cell lines. ns not significant; ∗∗∗∗p < 0.0001.

Article Snippet: Taqman gene expression assay probes were used to measure the mRNA levels of 194-1 (Thermo Fisher: Cat# Hs04231530_s1), 194-2 (Thermo Fisher: Cat# Hs04331541_s1), ALDH1A3 (Thermo Fisher: Cat# Hs00167476_m1), CD44 (Thermo Fisher: Cat# Hs05662929_s1), LYN (Thermo Fisher: Cat# Hs01015818_g1), WT1 (Thermo Fisher: Cat# Hs01103751_m1), CD133 (Thermo Fisher: Cat# Hs01009259_m1), Nestin (Thermo Fisher: Cat# Hs04187831_g1), OLIG1 (Thermo Fisher: Cat# Hs00907227_s1), SOX2 (Thermo Fisher: Cat# Hs04234836_s1), klf4 (Thermo Fisher: Cat# Hs00358836_m1), OCT4 (Thermo Fisher: Cat# Hs00999632_g1), FUT4 (Thermo Fisher: Cat# Hs01106466_s1), NCAM1 (Thermo Fisher: Cat# Hs00941830_m1), Nanog (Thermo Fisher: Cat# Hs02387400_g1), TAB2 (Thermo Fisher: Cat# Hs00248373_m1), CMYC (Thermo Fisher: Cat# Hs00153408_m1), DOCK5 (Thermo Fisher: Cat# Hs07287975_m1), FAS (Thermo Fisher: Cat# Hs00236330_m1), HFE (Thermo Fisher: Cat# Hs00373474_m1), LGALS3 (Thermo Fisher: Cat# Hs07288627_m1), MAL (Thermo Fisher: Cat# Hs00707014_s1), RHOF (Thermo Fisher: Cat# Hs00368032_m1), SOWAHA (Thermo Fisher: Cat# Hs00703106_s1), C21orf91 (Thermo Fisher: Cat# Hs05040994_s1), TRIM23 (Thermo Fisher: Cat# Hs01106626_m1).

Techniques: Luciferase, Reporter Assay, Inhibition, Expressing, Control, Clone Assay, Transfection, Activity Assay, Transduction, Marker

Journal: iScience

Article Title: miRNA-194-3p represses NF-κB in gliomas to attenuate iPSC genes and proneural to mesenchymal transition

doi: 10.1016/j.isci.2023.108650

Figure Lengend Snippet: TAB2 silencing transforms GSC/P phenotype by downregulating NF-κB and iPSC genes promoting adherent/M phenotype (A) Western blot analysis of the indicated proteins in U87-shTAB2 and U87-miR-194-3p isogenic cells. (B) TAB2 expression levels in OSU53-shTAB2 cell lines. Data are shown as mean ± SEM. Statistical significance was tested using two-tailed unpaired t-test. (C) Microscopic image showing the morphological features of isogenic shTAB2 cells. (D and E) Relative NF-κB activity in TAB2 and miR-194-3p isogenic cells ± competitive inhibitor measured using the NF-κB p65 Transcription Factor Assay Kit. Data are shown as mean ± SEM. Statistical significance was tested using two-tailed unpaired t-test. (F) Expression levels of P/M and iPSC marker in isogenic OSU53-shTAB2 cells. Data are shown as mean ± SEM. Statistical significance was tested using two-tailed unpaired t-test. (G) Western blot analysis of TAB2, NF-κB, and iPSC markers in OSU53-shTAB2 and OSU53-miR-194-3p isogenic cells. ns not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: Taqman gene expression assay probes were used to measure the mRNA levels of 194-1 (Thermo Fisher: Cat# Hs04231530_s1), 194-2 (Thermo Fisher: Cat# Hs04331541_s1), ALDH1A3 (Thermo Fisher: Cat# Hs00167476_m1), CD44 (Thermo Fisher: Cat# Hs05662929_s1), LYN (Thermo Fisher: Cat# Hs01015818_g1), WT1 (Thermo Fisher: Cat# Hs01103751_m1), CD133 (Thermo Fisher: Cat# Hs01009259_m1), Nestin (Thermo Fisher: Cat# Hs04187831_g1), OLIG1 (Thermo Fisher: Cat# Hs00907227_s1), SOX2 (Thermo Fisher: Cat# Hs04234836_s1), klf4 (Thermo Fisher: Cat# Hs00358836_m1), OCT4 (Thermo Fisher: Cat# Hs00999632_g1), FUT4 (Thermo Fisher: Cat# Hs01106466_s1), NCAM1 (Thermo Fisher: Cat# Hs00941830_m1), Nanog (Thermo Fisher: Cat# Hs02387400_g1), TAB2 (Thermo Fisher: Cat# Hs00248373_m1), CMYC (Thermo Fisher: Cat# Hs00153408_m1), DOCK5 (Thermo Fisher: Cat# Hs07287975_m1), FAS (Thermo Fisher: Cat# Hs00236330_m1), HFE (Thermo Fisher: Cat# Hs00373474_m1), LGALS3 (Thermo Fisher: Cat# Hs07288627_m1), MAL (Thermo Fisher: Cat# Hs00707014_s1), RHOF (Thermo Fisher: Cat# Hs00368032_m1), SOWAHA (Thermo Fisher: Cat# Hs00703106_s1), C21orf91 (Thermo Fisher: Cat# Hs05040994_s1), TRIM23 (Thermo Fisher: Cat# Hs01106626_m1).

Techniques: Western Blot, Expressing, Two Tailed Test, Activity Assay, Transcription Factor Assay, Marker

Journal: iScience

Article Title: miRNA-194-3p represses NF-κB in gliomas to attenuate iPSC genes and proneural to mesenchymal transition

doi: 10.1016/j.isci.2023.108650

Figure Lengend Snippet: TAB2 silencing enhances radiation sensitivity and prolongs survival of nude mice bearing intracranial tumors and graphical summary (A) Protein expression levels of TAB2 and NF-κB activation in T98G and OSU2 cell lines transduced with shTAB2. (B–D) Clonogenic assay comparing the radiosensitivity of NT and shTAB2 cell lines. Cells were fixed and stained 14 days following radiation. Data are shown as mean ± SEM. Statistical significance was tested using two-tailed unpaired t-test comparing the 6 Gy RER values of shTAB2 to NT in each cell line. The p value for each cell line was less than 0.01. (E) Kaplan-Meier curves demonstrating the survival of mice following intracranial implantation of U87-, OSU2-, and OSU53-shTAB2 isogenic cell lines. The p value was calculated using the two-sided log rank test. (F) H&E-stained coronal sections of tumors from nude mice bearing OSU53-shTAB2 isogenic cells. (G) Schematic summary representing the role of miR-194-3p in regulating PMT by inhibiting TAB2 and NF-κB activity. ns not significant; ∗∗p < 0.01.

Article Snippet: Taqman gene expression assay probes were used to measure the mRNA levels of 194-1 (Thermo Fisher: Cat# Hs04231530_s1), 194-2 (Thermo Fisher: Cat# Hs04331541_s1), ALDH1A3 (Thermo Fisher: Cat# Hs00167476_m1), CD44 (Thermo Fisher: Cat# Hs05662929_s1), LYN (Thermo Fisher: Cat# Hs01015818_g1), WT1 (Thermo Fisher: Cat# Hs01103751_m1), CD133 (Thermo Fisher: Cat# Hs01009259_m1), Nestin (Thermo Fisher: Cat# Hs04187831_g1), OLIG1 (Thermo Fisher: Cat# Hs00907227_s1), SOX2 (Thermo Fisher: Cat# Hs04234836_s1), klf4 (Thermo Fisher: Cat# Hs00358836_m1), OCT4 (Thermo Fisher: Cat# Hs00999632_g1), FUT4 (Thermo Fisher: Cat# Hs01106466_s1), NCAM1 (Thermo Fisher: Cat# Hs00941830_m1), Nanog (Thermo Fisher: Cat# Hs02387400_g1), TAB2 (Thermo Fisher: Cat# Hs00248373_m1), CMYC (Thermo Fisher: Cat# Hs00153408_m1), DOCK5 (Thermo Fisher: Cat# Hs07287975_m1), FAS (Thermo Fisher: Cat# Hs00236330_m1), HFE (Thermo Fisher: Cat# Hs00373474_m1), LGALS3 (Thermo Fisher: Cat# Hs07288627_m1), MAL (Thermo Fisher: Cat# Hs00707014_s1), RHOF (Thermo Fisher: Cat# Hs00368032_m1), SOWAHA (Thermo Fisher: Cat# Hs00703106_s1), C21orf91 (Thermo Fisher: Cat# Hs05040994_s1), TRIM23 (Thermo Fisher: Cat# Hs01106626_m1).

Techniques: Expressing, Activation Assay, Transduction, Clonogenic Assay, Staining, Two Tailed Test, Activity Assay

Journal: iScience

Article Title: miRNA-194-3p represses NF-κB in gliomas to attenuate iPSC genes and proneural to mesenchymal transition

doi: 10.1016/j.isci.2023.108650

Figure Lengend Snippet:

Article Snippet: Taqman gene expression assay probes were used to measure the mRNA levels of 194-1 (Thermo Fisher: Cat# Hs04231530_s1), 194-2 (Thermo Fisher: Cat# Hs04331541_s1), ALDH1A3 (Thermo Fisher: Cat# Hs00167476_m1), CD44 (Thermo Fisher: Cat# Hs05662929_s1), LYN (Thermo Fisher: Cat# Hs01015818_g1), WT1 (Thermo Fisher: Cat# Hs01103751_m1), CD133 (Thermo Fisher: Cat# Hs01009259_m1), Nestin (Thermo Fisher: Cat# Hs04187831_g1), OLIG1 (Thermo Fisher: Cat# Hs00907227_s1), SOX2 (Thermo Fisher: Cat# Hs04234836_s1), klf4 (Thermo Fisher: Cat# Hs00358836_m1), OCT4 (Thermo Fisher: Cat# Hs00999632_g1), FUT4 (Thermo Fisher: Cat# Hs01106466_s1), NCAM1 (Thermo Fisher: Cat# Hs00941830_m1), Nanog (Thermo Fisher: Cat# Hs02387400_g1), TAB2 (Thermo Fisher: Cat# Hs00248373_m1), CMYC (Thermo Fisher: Cat# Hs00153408_m1), DOCK5 (Thermo Fisher: Cat# Hs07287975_m1), FAS (Thermo Fisher: Cat# Hs00236330_m1), HFE (Thermo Fisher: Cat# Hs00373474_m1), LGALS3 (Thermo Fisher: Cat# Hs07288627_m1), MAL (Thermo Fisher: Cat# Hs00707014_s1), RHOF (Thermo Fisher: Cat# Hs00368032_m1), SOWAHA (Thermo Fisher: Cat# Hs00703106_s1), C21orf91 (Thermo Fisher: Cat# Hs05040994_s1), TRIM23 (Thermo Fisher: Cat# Hs01106626_m1).

Techniques: Recombinant, Protease Inhibitor, Transfection, Isolation, cDNA Synthesis, MTT Assay, Extraction, Transcription Factor Assay, Expressing, Plasmid Preparation, Reporter Assay, Staining, Negative Control, shRNA, Software

Journal: Cell

Article Title: A tissue injury sensing and repair pathway distinct from host pathogen defense

doi: 10.1016/j.cell.2023.03.031

Figure Lengend Snippet: (A) Schematic of two C57BL/6J Il24 −/− mouse strains generated by CRISPR-Cas9-mediated frameshift deletions within Il24 exon 2. Impairments of wound repair were indistinguishable between two loss-of- Il24- function strains, used interchangeably for experiments. (B) Sagittal sections of day-3 wounds from wild-type (WT) vs. Il24 null mice immunolabeled for p-STAT3. Note that p-STAT3 is still seen in Il24 null wounded epidermis (asterisk). Graphs show quantifications of the percentage of EpdSCs expressing p-STAT3 (upper), and the thickness of keratin 14 (KRT14 + ) progenitor layers (lower) (n = 5 mice per genotype). (C) Il20rb RNA-seq of FACS-purified cell populations from homeostatic skin and day-5 wounds (note: immune cells were only from day-5 wounds). TPM, transcripts per kilobase million (n = 5 mice). (D) Sagittal sections of day-5 wounds immunolabeled for KRT14 (epidermis), CD31 (endothelial cells), and labeled with 5-ethynyl-2′-deoxyuridine (EdU) (proliferation). Boxed regions are magnified in insets to better visualize EdU incorporation of S-phase cells (scale bars, 10 μm). Graphs show quantifications of percentage of EdU + cells in epidermis and dermis. For epidermis, quantifications were performed separately for the cells in the migrating zone (to the right of the wound site) and behind the migrating zone (to the left of the wound site) (n = 5 mice per genotype). (E) Left: quantifications of the percentages of migrating epidermis displaying adjacent CD31 + endothelial cells (top) and the percentages of the wound beds at day-5 and −7 post wounding that were repopulated with sprouting blood vessels (CD31 + cells) (middle and bottom). Mouse genotypes are as indicated (see ). Top and middle: WT: n = 5, Il24 Het: n = 6, Il24 −/− : n = 9 mice, one-way ANOVA, Tukey’s multiple comparisons test; bottom, WT: n = 5, Il20rb −/− :n =6 mice, two-tailed unpaired t test; dots in the graphs indicate data from individual mice. Right: Images of whole-mount immunofluorescence microscopy and 3D image reconstruction performed on day-5 wounds from WT vs. Il24 null mice (scale bars, 50 μm. Immunolabeling was for KRT14 [epidermis] and endomucin [blood vessels]) (n = 3 mice per genotype). (F) Sagittal sections of day-5 wounds immunolabeled for CD31 and PDGFRα (left), or for PDGFRα , collagen-I, and KRT14 (right). Asterisk (*) denotes a paucity of fibroblasts ( PDGFRα + ) and their deposition of collagen-I ECM in the dermis of Il24 −/− skin. The boxed region magnified in the color-coded insets shows additional Ki67 immunolabeling (Scale bars, 20 μm). Yellow arrows denote Ki67 + proliferating fibroblasts (Ki67 + PDGFR + ). Quantifications are of fibroblast amount ( PDGFRα intensity, upper) and collagen deposition (lower) (n = 5 per genotype). (G) Sagittal sections of day-5 wounds immunolabeled for p-STAT3 and KRT14. Percentage and number/area of p-STAT3 + dermal cells beneath the wound bed are quantified (n = 3 mice per genotype). (H) Left: sleeping beauty system used to generate epidermal-specific Il24 mRNA knockdown mice. Middle top: qRT-PCR of Il24 mRNA in FACS-purified EpdSCs from homeostatic and day-1 wounded skins from control (Ctrl) vs. sh Il24 mice (n = 5–6 mice for each genotype). Right: sagittal sections of day-5 wounds from control (Ctrl) vs. sh Il24 mice immunolabeled for CD31, KRT14 and labeled with EdU. Percentage of migrating epidermis adjacent to CD31 + capillaries is quantified in middle bottom panel (n = 6 mice per genotype). White dotted lines: epidermal-dermal border; wound site, red dotted line; epidermal migration direction, red arrow. DAPI, nuclei; scale bars except for boxed regions and whole mount: 100 μm. Data in (B)–(H) are presented as mean ± SEM. Dots in the graphs (E) and (H) indicate data from individual mice. Statistical significance was determined using two-tailed unpaired Student’s t tests in (D), (E; bottom panel), (F), (G), and (H); and using one-way ANOVA, Tukey’s multiple comparisons test in (B) and (E; top two panels); **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; and ns, not significant. See also – .

Article Snippet: Rat monoclonal anti-Endomucin antibody , Santa Cruz Biotechnology , Cat# sc-65495; RRID:AB_2100037.

Techniques: Generated, CRISPR, Immunolabeling, Expressing, RNA Sequencing, Purification, Labeling, Two Tailed Test, Immunofluorescence, Microscopy, Knockdown, Quantitative RT-PCR, Control, Migration

Journal: Cell

Article Title: A tissue injury sensing and repair pathway distinct from host pathogen defense

doi: 10.1016/j.cell.2023.03.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rat monoclonal anti-Endomucin antibody , Santa Cruz Biotechnology , Cat# sc-65495; RRID:AB_2100037.

Techniques: Virus, Recombinant, SYBR Green Assay, Lysis, Extraction, Protease Inhibitor, CRISPR, Saline, Western Blot, Clinical Proteomics, Purification, RNA Library Preparation, Multiplex Assay, cDNA Synthesis, Bicinchoninic Acid Protein Assay, Staining, Imaging, Sequencing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Software, Microscopy, Sterility

Journal: Nature Communications

Article Title: Human Cyclophilin B forms part of a multi-protein complex during erythrocyte invasion by Plasmodium falciparum

doi: 10.1038/s41467-017-01638-6

Figure Lengend Snippet: Cyclophilin B is a novel erythrocyte receptor for P. falciparum merozoite binding. a Parameters highlighting length, molecular weight (MW), Iso-electric point (pI), number of cysteines and grand average of hydropathicity (GRAVY) of the identified host-pathogen interacting protein partners Cyclophilin B (CypB) and PfRhopH3-C. b Bacterial two-hybrid assay between identified host-pathogen interacting partners. Streaks of the identified prey protein from the bacterial two-hybrid experiment between PfRhopH3-C and human lung cDNA library on X-gal indicator plate. All streaks are labeled to represent genes cloned in pBTnn and pTRGnn. CFP10-pTRGnn/empty pBTnn is the negative control; CFP10pTRGnn/ESAT6pBTnn is the positive control. c Liquid β-galactosidase assay to measure relative enzymatic activity of co-transformant pairs. Relative Miller’s units (M.U.) of RhopH3-CpBTqq/CYPBpTRGqq, CFP10pTRGnn/ESAT6pBTnn (positive control) and CFP10-pTRGnn/empty pBTnn (negative control) were plotted. The graph is the average of three independent assays with error bars representing the standard deviation; all values were tested for significance using a two-tailed unpaired Student’s t -test with Welch’s correction. ** P < 0.01, *** P < 0.001. d Localization of CypB on the RBC surface. Human erythrocytes were incubated with primary anti-CypB monoclonal antibody (mouse) followed by secondary alexa-fluor 488 conjugated goat anti-mouse IgG antibody (1:200) and confocal microscopy. e Binding of CypB on the merozoite surface. Merozoites were incubated with 25 µM recombinant CypB for 2 h followed by incubation with primary anti-CypB monoclonal antibody (mouse). The Merozoites were stained with alexa-fluor 488 conjugated goat anti-mouse IgG antibody (1:200; green) against primary antibody followed by confocal microscopy. Scale bar = 5 µm

Article Snippet: Recombinant proteins Cyclophilin B (Catlog: 11004-H08H-100) and Basigin (BSG/CD147, Catlog: 10186-H08H-100) were obtained commercially from Sino Biological Inc., China (Supplementary Fig. ).

Techniques: Binding Assay, Molecular Weight, Two Hybrid Assay, cDNA Library Assay, Labeling, Clone Assay, Negative Control, Positive Control, Activity Assay, Standard Deviation, Two Tailed Test, Incubation, Confocal Microscopy, Recombinant, Staining

Journal: Nature Communications

Article Title: Human Cyclophilin B forms part of a multi-protein complex during erythrocyte invasion by Plasmodium falciparum

doi: 10.1038/s41467-017-01638-6

Figure Lengend Snippet: PfRhopH3 protein is a ligand for Cyclophilin B protein during merozoite binding. a Binding of Cyclophilin B on Merozoite surface. Binding shows binding of CypB on merozoite surface without treatment with any antibody. anti-Rap2b + CypB and anti-RhopH3-C + CypB show binding after treatment with anti-Rap2b and anti-RhopH3 antibody respectively. Intensity for each merozoite is mentioned at the top. Images with histograms represent only the intensity of the bound CypB protein. Anti-RhopH3-C antibody interferes with binding of CypB on merozoites, signifying that PfRhopH3 is a ligand for host Cyclophilin B. Scale bar = 5 µm. b Graph adapted from Supplementary Table showing the average of binding intensity of CypB on 10 different merozoites, with error bars representing the standard deviation. All values were tested for significance using a two-tailed unpaired Student’s t -test. ** P < 0.01

Article Snippet: Recombinant proteins Cyclophilin B (Catlog: 11004-H08H-100) and Basigin (BSG/CD147, Catlog: 10186-H08H-100) were obtained commercially from Sino Biological Inc., China (Supplementary Fig. ).

Techniques: Binding Assay, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: Human Cyclophilin B forms part of a multi-protein complex during erythrocyte invasion by Plasmodium falciparum

doi: 10.1038/s41467-017-01638-6

Figure Lengend Snippet: Protein−protein interaction assays confirm the interaction between Cyclophilin B and PfRhopH3-C. a ELISA-based assay confirming the interaction between CypB and PfRhopH3-C. 200 ng of PfRhopH3-C protein was coated on ELISA plates. CypB was overlaid on subset of wells in increasing concentrations as shown, and detected by anti-CypB (rabbit polyclonal) antibody followed by HRP conjugated secondary anti-rabbit antibody. A non-specific antibody ((nsAb) anti-CFP10 rabbit polyclonal) was used as the antibody control. Negative control for this ELISA experiment is shown in Supplementary Fig. . Each bar represents the mean ± S.D. for triplicate experiment. b Far-western analysis. PfRhopH3-C was run on SDS-PAGE and transferred onto PVDF membrane and following denaturation and renaturation, incubated with CypB protein. CypB protein (indicated by arrow) was detected by anti-CypB (rabbit polyclonal) antibody. Mycobacterial protein HBHA was used as the negative control protein. c Co-immunoprecipitation of PfRhoph3-C with CypB and PfRh5. Antibody coupling resin bound with CypB and PfRh5 antibodies was incubated with CypB:PfRhopH3-C and PfRh5:PfRhopH3-C mixtures, respectively. Eluted samples were run on SDS-PAGE followed by western blotting. Detection of protein PfRhopH3-C (indicated by arrow) was carried out using anti-PfRhopH3 (rabbit polyclonal) antibody (1:5000). CFP10 was used as the negative control protein. d Surface plasmon resonance analysis of CypB and PfRhopH3-C interaction. The indicated concentrations of purified PfRhopH3-C were injected over immobilized CypB, and biophysical parameters were derived from a 1:1 binding model. RU, response units. K D value for this interaction was 1.6 × 10 −7 M. e Binding and co-localization of PfRHopH3-C on the RBC surface. Uninfected RBCs were incubated with 20 μM PfRhopH3-C and stained with anti-RhopH3-C antibody (rabbit) and anti-CypB antibody (mouse). Subsequently, secondary anti-rabbit alexa-flour 594 antibody and anti-mouse alexa-flour 488 antibody were used to detect the co-localization of the proteins. Scale bar = 5 µm

Article Snippet: Recombinant proteins Cyclophilin B (Catlog: 11004-H08H-100) and Basigin (BSG/CD147, Catlog: 10186-H08H-100) were obtained commercially from Sino Biological Inc., China (Supplementary Fig. ).

Techniques: Enzyme-linked Immunosorbent Assay, Negative Control, Western Blot, SDS Page, Incubation, Immunoprecipitation, SPR Assay, Purification, Injection, Derivative Assay, Binding Assay, Staining

Journal: Nature Communications

Article Title: Human Cyclophilin B forms part of a multi-protein complex during erythrocyte invasion by Plasmodium falciparum

doi: 10.1038/s41467-017-01638-6

Figure Lengend Snippet: Cyclophilin B interacts with Basigin (CD147/BSG), a critical merozoite invasion receptor on RBCs. a Co-localization of CypB and BSG on the surface of the RBC. RBCs were fixed and incubated with anti-CypB (mouse monoclonal) and anti-BSG (rabbit polyclonal) antibodies. The cells were stained with fluorochrome-conjugated secondary antibodies against CypB (green) and BSG (red) followed by confocal microscopy. CypB and BSG co-localized on the RBC surface with a Pearson’s coefficient of 0.53. b Merozoites were incubated with 25 µM of BSG protein for 2 h and detection was carried out using anti-CD147 antibody (rabbit polyclonal) and secondary fluorochrome-conjugated (red), followed by confocal microscopy. c CypB and BSG bind together on the merozoite surface. Merozoites were incubated with CypB and BSG proteins (25 µM each) for 2 h and binding was detected using anti-CypB (mouse monoclonal) and anti-BSG (rabbit polyclonal) antibodies followed by secondary antibodies and confocal microscopy. Supplementary Fig. illustrates in vitro interaction assays between CypB and BSG. Scale bar = 5 µm

Article Snippet: Recombinant proteins Cyclophilin B (Catlog: 11004-H08H-100) and Basigin (BSG/CD147, Catlog: 10186-H08H-100) were obtained commercially from Sino Biological Inc., China (Supplementary Fig. ).

Techniques: Incubation, Staining, Confocal Microscopy, Binding Assay, In Vitro

Journal: Nature Communications

Article Title: Human Cyclophilin B forms part of a multi-protein complex during erythrocyte invasion by Plasmodium falciparum

doi: 10.1038/s41467-017-01638-6

Figure Lengend Snippet: Identification of a de novo peptide that binds Cyclophilin B. a Bacterial two-hybrid assay to identify de novo interacting partner against CYPB from dicodon library. Streaks of bacterial two-hybrid experiments between DIEL-pBTnn and CYPBpTRGqq on X-gal indicator plate. All streaks are labeled to represent genes cloned in pBTnn and pTRGnn. CFP10pTRGnn/Emptyp BTnn and CFP10pTRGnn/ESAT6pBTnn signify the negative and positive controls, respectively. b Liquid β-galactosidase assay to estimate the relative enzymatic activity in Miller’s unit (M.U.) of the interaction between identified de novo dicodon library interacting partner (CDP3) and CYPB. The graph is the average of three independent assays with error bars representing the standard deviation. All values were tested for significance using a two-tailed unpaired Student’s t -test with Welch’s correction. *** P < 0.001. c , d Bacterial three-hybrid assay to monitor the disruption of PfRhopH3-C/CypB interaction by CDP3 in vivo. X-Gal indicator plate without l -arabinose and X-gal indicator plate with l -arabinose. Test streaks: Triple co-transformants containing PfRhopH3CpBTqq, CYPBpTRGqq, and CDP3pMTSA; control streaks: triple co-transformants containing PfRhopH3-CpBTqq, CYPBpTRGqq, and empty pMTSA. e l -arabinose gradient liquid β-galactosidase assay. Relative β-galactosidase activity Miller’s unit (M.U.) of the triple co-transformants containing PfRhopH3-CpBTqq, CYPBpTRGqq and empty pMTSA (red line, control) and triple co-transformants containing PfRhopH3CpBTqq, CYPBpTRGqq, and CDP3pMTSA (black line, test), is plotted against a range of l -arabinose concentrations. The graph is the average of three independent assays and the standard deviation is represented as the error bars. Multiple unpaired t -tests to compare enzyme activity of each triple co-transformant across individual l -arabinose concentrations were used. Statistical significance was determined using the unpaired t -test with Welch’s correction. ** P < 0.01 was considered significant. f Western blot of triple co-transformants R1 E. coli whole-cell lysates shown in e , in order to analyze the concomitant expression of CDP3 protein with increasing concentrations of l -arabinose

Article Snippet: Recombinant proteins Cyclophilin B (Catlog: 11004-H08H-100) and Basigin (BSG/CD147, Catlog: 10186-H08H-100) were obtained commercially from Sino Biological Inc., China (Supplementary Fig. ).

Techniques: Two Hybrid Assay, Labeling, Clone Assay, Activity Assay, Standard Deviation, Two Tailed Test, Hybrid Assay, In Vivo, Western Blot, Expressing

Journal: Nature Communications

Article Title: Human Cyclophilin B forms part of a multi-protein complex during erythrocyte invasion by Plasmodium falciparum

doi: 10.1038/s41467-017-01638-6

Figure Lengend Snippet: Treatment of human RBCs with CDP3 and Cyclosporin A (CsA) suggests Cyclophilin B plays a crucial role in P. falciparum invasion of host RBCs. Invasion Assay using CDP3: a Invasion inhibition of P. falciparum strains 3D7 and Dd2 by CDP3 into culture. Purified recombinant CDP3 protein (1–25 µM) was added to mature schizont stage parasite culture and the parasitaemia estimated after 40 h using flow cytometry. The data represent an average of three independent experiments each performed in duplicate. Invasion observed in control culture was taken as 100%. The CDP3 buffer components did not affect parasite invasion. b Similarly, Uninfected RBCs were treated with CDP3 (10, 25, 50 µM) for 4 h at RT followed by 3D7 merozoites infection. Parasitemia was analyzed using FACS. c − e Dose dependent inhibition of invasion by CsA treatment of RBC: Uninfected BCs treated with CsA (2.5–100 µM) followed by 3D7 merozoites infection. Invasion was reduced by ~80%. Similar results were obtained by using different strains of P. falciparum including Dd2, HB3 and 7G8. CsA (12.5–50 µM) added in culture at late schizont stage; parasitemia was estimated after 40 h. 80–90% inhibition of invasion was observed. Statistical significance was determined using the unpaired t -test with Welch’s correction. ** P < 0.01 was considered significant. Each bar represents the mean ± S.D. for three independent biological replicates

Article Snippet: Recombinant proteins Cyclophilin B (Catlog: 11004-H08H-100) and Basigin (BSG/CD147, Catlog: 10186-H08H-100) were obtained commercially from Sino Biological Inc., China (Supplementary Fig. ).

Techniques: Invasion Assay, Inhibition, Purification, Recombinant, Flow Cytometry, Infection

Journal: The EMBO Journal

Article Title: EPDR1 promotes PD-L1 expression and tumor immune evasion by inhibiting TRIM21-dependent ubiquitylation of IkappaB kinase-β

doi: 10.1038/s44318-024-00201-6

Figure Lengend Snippet: ( A , B ) Plasmids expressing human YAP5SA (2 mg/kg) plus RFP control (2 mg/kg) or mEPDR1 (2 mg/kg), together with plasmids expressing PB transposase (0.8 mg/kg), were delivered into mice by hydrodynamic injection ( n = 6 male mice per group). Liver tumors were analyzed 110 days after injection. Photographs show livers ( A ) and tumor numbers ( B ) were determined. Data were presented as the mean ± SEM. ( C ) Dimensionality reduction and visualization based on T-distributed stochastic neighbor embedding (t-SNE) analysis of a subset of mouse liver immunocytes from the indicated group in ( A ). ( D ) Statistical difference analysis for immune cell subsets was obtained from dimensionality reduction analysis in panel ( C ). n = 5 male mice per group and data were presented as the mean ± SD. ( E ) Flow cytometry analysis of the ratio of the immune co-suppressive molecules (PD-1, TIM-3) and immune effector molecules (IFNγ, GzmB) positive cells in liver CD8 + T cells from the indicated group in ( A ). n = 6 male mice per group and data were presented as the mean ± SD. ( F ) Schema of coculture of human CD8 + T cells with HepG2 cells expressing Flag-EV or Flag-EPDR1. ( G ) Flow cytometry analysis of the ratio of immunosuppressive molecules (PD-1, TIM-3) and immune effector molecules (IFNγ, GzmB) positive cells in CD8 + T cells after coculture with the indicated tumor cells. n = 3 independent experiments and the data were presented as the mean ± SD. ( H – J ) Hepa 1-6 cells stably expressing Flag-EV or Flag-mEPDR1 were injected subcutaneously into C57BL/6 J mice ( n = 6 male mice per group), and α-CD8 (4 mg/kg) neutralizing antibody was injected intraperitoneally four times (twice a week starting at 10 days after inoculation) to block CD8 + T cells and IgG2b was used as control. Tumor size was measured starting at 10 days after inoculation. Photographs show xenografts ( H ), growth curves ( I ), and relative tumor burdens ( J ) determined at the end of the experiment (day 25). Data were presented as the mean ± SEM. Data information: Statistical significance was determined by two-way ANOVA ( D , I ), one-way ANOVA ( J ), and two-tailed unpaired Student’s t -test ( B , E , G ). .

Article Snippet: InVivoMAb rat IgG2b isotype control , BioXcell , BE0090.

Techniques: Expressing, Control, Injection, Flow Cytometry, Stable Transfection, Blocking Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: EPDR1 promotes PD-L1 expression and tumor immune evasion by inhibiting TRIM21-dependent ubiquitylation of IkappaB kinase-β

doi: 10.1038/s44318-024-00201-6

Figure Lengend Snippet: ( A ) qRT-PCR analysis of the mRNA levels of a series of immune-related molecules in HepG2 cells with EPDR1 knockdown. Data were presented as the mean ± SD of three independent experiments ( n = 3). ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 compared with NTC control. ( B ) Flow cytometry analysis of the membrane-bound PD-L1 in HepG2 cells expressing Flag-EV and Flag-EPDR1. Data were presented as the mean ± SD of three independent experiments ( n = 3). ( C ) Flow cytometry analysis of the membrane-bound PD-L1 in HepG2 cells with EPDR1 knockdown. Data were presented as the mean ± SD of three independent experiments ( n = 3). ( D ) Histogram of the KEGG enrichment analysis results for differentially expressed genes between HepG2 cells expressing shEPDR1 and those expressing shNTC. P value was computed using the one-sided Fisher’s exact test and corrected for multiple hypothesis testing using a false discovery rate (FDR). ( E ) Western blotting analysis of the protein levels of PD-L1 and p65 in whole cell lysates and nuclear fractions in HepG2 cells expressing shNTC and shEPDR1 with GAPDH and Lamin B as loading controls, respectively. ( F ) Western blotting analysis of the protein levels of PD-L1 and p65 in HepG2 cells expressing shNTC and shEPDR1. Cells were treated with the NF-κB agonist TNFα (100 nM) or vehicle control 6 h before sample collection, and β-actin was used as a loading control. ( G ) Western blotting analysis of the protein levels of PD-L1 and p65 in HepG2 cells expressing Flag-EV and Flag-mEPDR1. Cells were treated with the NF-κB inhibitor BAY11-7082 (5 μM) or vehicle control 6 h before sample collection, and β-actin was used as a loading control. ( H ) ChIP analysis of the occupancy of p65 on the PD-L1 promoters in HepG2 cells expressing shNTC and shEPDR1. Data were presented as the mean ± SD of three independent experiments ( n = 3). ( I ) Scatter diagram showing the potential interactors of EPDR1. ( J ) Co-IP assay showing the protein interaction between EPDR1 and TRIM21. HepG2 cells were infected with lentivirus carrying Flag-EV or Flag-EPDR1 together with HA-TRIM21 plasmids. Cell lysates were immunoprecipitated with an anti-Flag antibody, followed by Western blotting analysis with antibodies against Flag and HA tags. ( K ) Pull-down assay showing the protein interaction between GST-TRIM21 and His-EPDR1. GST-tagged TRIM21 and 6 × His-tagged EPDR1 proteins were purified from E. coli and incubated in vitro, followed by Western blotting analysis with antibodies against TRIM21 or EPDR1. The red asterisks indicate the target bands. ( L ) Co-IP assay showing the protein interaction between TRIM21 and IKBKB in the indicated genotypes. HepG2 cells expressing empty vector or EPDR1 were infected with lentivirus carrying Flag-IKBKB with HA-EV or HA-TRIM21 plasmids. Cell lysates were immunoprecipitated with an anti-HA antibody, followed by Western blotting analysis with antibodies against IKBKB, EPDR1, and HA tags. ( M ) Pull-down assay showing the protein interaction of GST-TRIM21 and His-EPDR1 or His-IKBKB. 6× His-tagged EPDR1 and IKBKB proteins were purified from E. coli and incubated with purified GST-tagged TRIM21 alone or together in vitro, followed by Western blotting analysis with antibodies against TRIM21, EPDR1 or IKBKB. The red asterisks indicate the target bands. ( N ) HEK293T cells expressing Flag-IKBKB and hemagglutinin-tagged ubiquitin (HA-Ub) were co-transfected with EV, EPDR1, TRIM21 plasmids alone, or EPDR1 plus TRIM21 for 48 h and treated with 10 mM 3-MA for 8 h before collection. Immunoprecipitation was performed using anti-Flag antibody or IgG using the above cells. Polyubiquitination of Flag-IKBKB was detected by Western blotting. ( O ) Western blotting analysis of the protein levels of PD-L1, IKBKB, and p65 in whole cell lysates and nuclear fractions in HepG2 cells expressing Flag-EPDR1 and/or HA-TRIM21, with GAPDH and Lamin B, respectively, as loading controls. Data information: Statistical significance was determined by two-way ANOVA ( A , H ), two-tailed unpaired Student’s t -test ( B ), and one-way ANOVA ( C ). .

Article Snippet: InVivoMAb rat IgG2b isotype control , BioXcell , BE0090.

Techniques: Quantitative RT-PCR, Knockdown, Control, Flow Cytometry, Membrane, Expressing, Western Blot, Co-Immunoprecipitation Assay, Infection, Immunoprecipitation, Pull Down Assay, Purification, Incubation, In Vitro, Plasmid Preparation, Ubiquitin Proteomics, Transfection, Two Tailed Test

Journal: The EMBO Journal

Article Title: EPDR1 promotes PD-L1 expression and tumor immune evasion by inhibiting TRIM21-dependent ubiquitylation of IkappaB kinase-β

doi: 10.1038/s44318-024-00201-6

Figure Lengend Snippet: ( A – C ) Hepa 1-6 cells stably expressing Flag-EV and Flag-mEPDR1 were injected subcutaneously into C57BL/6J mice ( n = 6 male mice per group), and α-PD-L1 (6 mg/kg) neutralizing antibody was injected intraperitoneally four times (twice a week starting at 10 days after inoculation) to block PD-L1 and IgG2b was used as control. Tumor size was measured starting at 10 days after inoculation. Photographs show xenografts ( A ), growth curves ( B ), and tumor weight ( C ) determined at the end of the experiment (day 25). Data were presented as the mean ± SEM ( B ) and mean ± SD ( C ), respectively. ( D ) Flow cytometry analysis of ratio of immunosuppressive molecules (PD-1, TIM-3) positive cells in tumor CD8 + T cells from the indicated group in ( A ). Data were presented as the mean ± SD. ( E ) Flow cytometry analysis of the ratio of immune effector molecules (IFNγ, Granzyme B) positive cells in tumor CD8 + T cells from the indicated groups in ( A ). Data were presented as the mean ± SD. ( F – H ) Hepa 1–6 cells stably expressing Flag-EV and Flag-mEPDR1 were injected subcutaneously into C57BL/6J mice ( n = 6 male mice per group). BAY11-7082 was used to inhibit the NF-Κb pathway, and the vehicle was used as a control. Tumor size was measured starting at 10 days after inoculation. Photographs show xenografts ( F ), growth curves ( G ), and final tumor weight ( H ) determined at the end of the experiment (day 25). Data were presented as the mean ± SEM ( G ), and mean ± SD ( H ), respectively. ( I ) Flow cytometry analysis of the ratio of immunosuppressive molecules (PD-1, TIM-3) positive cells in tumor CD8 + T cells from the indicated group in ( F ). Data were presented as the mean ± SD. ( J ) Flow cytometry analysis of the ratio of immune effector molecules (IFNγ, GzmB) positive cells in tumor CD8 + T cells from the indicated group in ( F ). Data were presented as the mean ± SD. Data information: Statistical significance was determined by two-way ANOVA ( B , G ) and one-way ANOVA ( C – E , H – J ). .

Article Snippet: InVivoMAb rat IgG2b isotype control , BioXcell , BE0090.

Techniques: Stable Transfection, Expressing, Injection, Blocking Assay, Control, Flow Cytometry

Journal: The EMBO Journal

Article Title: EPDR1 promotes PD-L1 expression and tumor immune evasion by inhibiting TRIM21-dependent ubiquitylation of IkappaB kinase-β

doi: 10.1038/s44318-024-00201-6

Figure Lengend Snippet: Reagents and tools table

Article Snippet: InVivoMAb rat IgG2b isotype control , BioXcell , BE0090.

Techniques: Recombinant, Purification, Sequencing, Control, Modification, Cell Stimulation, Ligation, Cloning, cDNA Synthesis, SYBR Green Assay, Software