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  • 80
    Millipore mouse ikkβ gene sequence
    Pharmacological inhibition of IKK β inhibits adipocyte differentiation. (A) Oil red O staining of 3T3-L1 cells induced by differentiation medium or medium containing <t>IKKβ</t> inhibitor BMS-345541 at indicated concentrations. (B) Analysis of IKKβ, Smurf2, and adipogenic genes in 3T3-L1 cells treated with control or 10 µM BMS-345541 for 48 h by QPCR ( n = 3–5). (C) 3T3-L1 cells were treated with vehicle control or 10 µM BMS-345541 for 48 h before incubating with vehicle control or 100 nM PS-341 for 4 h. β-catenin was immunoprecipitated with anti–β-catenin antibodies, and then probed with antiubiquitin monoclonal antibodies. The whole-cell lysates were probed with anti–β-catenin antibodies as an internal control. (D) Western blot analysis of nuclear β-catenin levels in 3T3-L1 cells treated with vehicle control or 10 µM BMS-345541 for 48 h. Nuclear proteins were probed with anti-Histone H3 antibodies as an internal control. (E) Oil red O staining of 3T3-L1 cells induced by differentiation medium or medium containing IKKβ inhibitor sodium salicylate at indicated concentrations. Similar results were obtained from at least three independent experiments. All data are mean ± SD. **, P
    Mouse Ikkβ Gene Sequence, supplied by Millipore, used in various techniques. Bioz Stars score: 80/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher mouse cx47 coding sequence
    Immunoblots showing co-IP of ZO-1 and <t>Cx47</t> from various brain regions and from Cx47-transfected HeLa cells. (A) Homogenates of brain regions were taken for IP with anti-ZO-1 antibody and immunoblots of precipitates were probed with anti-Cx47 antibody.
    Mouse Cx47 Coding Sequence, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    GE Healthcare short hairpin sequences targeting clusterin
    TGF-β 1 associates with areas of decreased <t>clusterin</t> expression in fibrotic lung and down-regulates fibroblast clusterin mRNA and protein expression in vitro . Serial sections prepared from IPF lung (n = 3) were stained immunohistochemically to localize TGF-β 1 staining (( A ), red/brown, nuclei blue) and clusterin (( B ), red/brown, nuclei blue) in fibroblastic foci. ( A,B ) Representative images of immunohistochemical staining suggest that TGF-β 1 localizes to ECM, fibroblasts and macrophages, whilst staining for clusterin is weak or undetectable. ( C ) In vitro analysis of clusterin mRNA levels in TGF-β 1 stimulated (40 pM) human lung fibroblasts was performed via qRT-PCR and shows a time-dependent down-regulation of clusterin expression that was maximal at 24–48 h (n = 3) in response to TGF-β 1 . Clusterin protein levels were also down-regulated in response to TGF-β 1 (40 pM) compared to control at 24 h and 48 h as demonstrated by western blotting at 24 h ( D ) and immunofluorescent staining at 48 h (E, red, nuclei - blue). ( F ) Semi-quantitative analysis of fluorescent signal of panel E: clusterin signal (pixel intensity) was normalized to cell numbers per visual field and compared to control (n = 6). Full-length western blots are presented in Supplementary Figure e4 . Different cell populations/structures are indicated by arrows; f - fibroblast-like cell, m - macrophage, he - hyperplastic epithelial cell, ecm – extracellular matrix. * P
    Short Hairpin Sequences Targeting Clusterin, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 88/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Vical lulohya sequence
    Characterization of <t>LuloHya.</t> Hyaluronidase activity is female specific in Lu . longipalpis . (A) SGE from 10 male Lu . longipalpis did not show any cleavage of HA in the turbidimetric assay, expressed as the % of remaining HA. On the contrary, SGE from 1 female and 10 nM recombinant LuloHya exhibited remarkable hyaluronidase activity. Multiple comparisons were done by one-way ANOVA (****: p
    Lulohya Sequence, supplied by Vical, used in various techniques. Bioz Stars score: 87/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    OriGene shrna sequence targeting mt1 mmp
    <t>MT1-MMP</t> overexpression inhibited the protrusive morphology of MDA-MB 231 breast cancer cells in 3D culture. a ( top ) MDA-MB 231 MT1-MMP cells were embedded in Matrigel and imaged every day for 5 days at 10× magnification. Shown are representative fields of view of each cell line at day 5 and a respective inset. Red arrow shows a portion of the protrusive network MDA-MB 231 cells form in 3D culture. White arrows show MDA-MB 231 MT1-MMP cell colonies that have retained circularity after 5 days in 3D culture. Scale bars = 100 μm. ( bottom ) Five z-stacks per cell line were acquired every day for 5 days and disseminations and protrusions were quantified per colony for each cell line. b Representative 3D volume views of immunofluorescence analysis after MDA-MB 231 MT1-MMP cells were embedded in Matrigel for 5 days. Samples were imaged using confocal microscopy at 60× magnification and are displayed as overlays showing MT1-MMP signal ( green ), DAPI ( blue ) and Alexa633 phalloidin ( red ) channels. Scale bars = 100 μm. Red arrow shows protrusive MDA-MB 231 cells, whereas green arrows show circular colonies in MDA-MB 231 MT1-MMP cell lines that are positive for MT1-MMP protein signal. Scale bars = 100 μm
    Shrna Sequence Targeting Mt1 Mmp, supplied by OriGene, used in various techniques. Bioz Stars score: 87/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher wri1 amino acid sequence
    Kinase Activity Is Necessary for KIN10-Dependent <t>WRI1</t> Degradation.
    Wri1 Amino Acid Sequence, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher t cadherin sense sequence
    Lack of <t>T-cadherin</t> mRNA in the developing heart of mouse embryos at the E8.75–E10.5 stages ( 1 , 2 , 3 ). Arrows and the selected area indicate the developing heart region. Magnification of 5× ( 1 , 2 ) and 6× ( 3 )
    T Cadherin Sense Sequence, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Thermo Fisher human npc2 sequence
    <t>NPC2</t> deficiency confers fibroblast activation. A , expression of activated fibroblast markers as probed by real-time RT-PCR is shown. IL-6 and IL-1β secretion from non-elicited normal and NPC2-null human fibroblasts as measured by ELISA is shown.
    Human Npc2 Sequence, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 81/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    The Jackson Laboratory endogenous lgr5 regulatory sequences
    Lineage tracing of cells with <t>Lgr5</t> transcriptional activity in H felis –infected Lgr5-Cre;H + /K + -Nog;Rosa26-Tom mice. 1-2-month old Lgr5-Cre;H + /K + -Nog;Rosa26-tdTom mice were treated with one i.p. injection of tamoxifen (0.1 mg/g body weight) and inoculated with H. felis two months post tamoxifen. Animals were analyzed 3 months after inoculation, and 5 months after tamoxifen injection. Gastric paraffin sections of the lesser curvature of H felis –infected Lgr5-Cre;H + /K + -Nog;Rosa26-tdTom mice were stained with anti-tomato (red fluorescent protein) primary antibodies and Alexa 555–conjugated secondary antibodies together with anti-IF antibodies and Alexa 488–conjugated secondary antibodies (IF/Tom), Alexa 488–conjugated GSII (GSII/Tom), Alexa 488–conjugated Ulex europaeus agglutinin 1 (UEA1), anti-CD44 and anti-CD44v9 primary antibodies and Alexa 488–conjugated secondary antibodies (CD44/Tom and CD44v 9/Tom), and anti-BrdU primary antibodies and Alexa 488–conjugated secondary antibodies (BrdU/Tom). Scale bars : 50 μm. Similar results were observed in at least 5 other H felis –infected Lgr5-Cre;H + /K + -Nog;Rosa26-tdTom mice except for the experiments with the anti-CD44v9 antibodies that were repeated in 1 other mouse.
    Endogenous Lgr5 Regulatory Sequences, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 80/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Thermo Fisher full length sequence verified human smpdl3a open reading frame
    <t>SMPDL3A</t> expression in cholesterol-loaded human monocyte-derived macrophages. A, SMPDL3A mRNA expression. HMDM from five individual healthy donors were incubated for 48 h in RPMI 1640 medium containing 10% (v/v) LPDS ± AcLDL (50 μg/ml),
    Full Length Sequence Verified Human Smpdl3a Open Reading Frame, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Thermo Fisher fat1 sequence
    Identification of contraction-independent FSHD patients carrying deletions of an intronic <t>FAT1</t> enhancer. ( A ) View of the Human genomic FAT1 locus focusing on an area including FAT1 exons 17-18-19. The lower image is a USCC browser based screen-copy image showing a track displaying ENCODE enhancer and promoter associated histone mark (H3K4me1) on 8 cell lines. ( B ) Positions of copy number variants identified in 5 FSHD patients by CGH and positioned on the genome by CGHweb analysis. Patients are identified with a specific number, and their characteristics are available in the Table S1 . The deletion span varies from deletions restricted to intron 17 to deletions spanning over intron 17, exon 17 and intron 16, including a ENCODE-putative enhancer visible through genomic browsers. ( C ) Copy number validation of the deletion by qPCR. The three graphs show the relative amounts of PCR fragments obtained using primers couples 1–2 (exon 17), 2–3 (enhancer intron 16) and 4–5 (exon 16)), in a group of 40 healthy controls (blue area), a group of 10 FSHD1 patients (red area), and a group of 19 contraction-independent patients (c.i.FSHD). All data were normalized using an unrelated genomic fragment (Adora) as internal control, and one of the control DNAs (number 21) was used as the reference DNA (where all values are set to 1). A cut-off of 0.75 has been set. Individuals in which the relative value is lower than the cut-off are considered as having lowered copy numbers (indicated as loss). Information on each patient (regarding clinical and genetic diagnostic) are available in the Table S1 . ( D ) The distribution of CNVs corresponding to loss CNV (seen as red) is shown in controls and in FSHD groups (all together, or FSHD1 and c.i.FSHD separately) for each of the three spots considered individually (top three graphs) or considered together (bottom plot, where loss represents the number of cases having a loss for at least one of the three spots). The cases where a significant link (as measured by X 2 or Fischer tests) are indicated with one or two stars (* for p
    Fat1 Sequence, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Research Genetics Inc p205 sequences
    Treatment of membranes with phalloidin increases the sedimentability of <t>p205,</t> as well as actin. ( A ) Neutrophil plasma membranes treated without (−) or with (+) phalloidin were solubilized in TEB and fractionated on 20–55% sucrose gradients. The initial membrane extract ( Load ) and gradient fractions (lanes 1–17 ) were analyzed for the presence of cytoskeletal proteins as described in Materials and Methods. Positions of calibration standards ( 9S , 19S , and 30S ) are indicated. ( B ) Phalloidin- induced shift in the sedimentability of p205. Average distribution of p205 in gradient fractions, expressed as a percent of the total F-actin binding at 205 kD on F-actin blot overlays ( n = 3). Similar results were observed when blot strips were re-probed with anti-pepA antibody, indicating that a single 205-kD F-actin–binding polypeptide is present in the 13S, 26S, and 30S complexes.
    P205 Sequences, supplied by Research Genetics Inc, used in various techniques. Bioz Stars score: 79/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore insm1 sequence
    The number of OE progenitors in the cell cycle or S-phase, as well as cell cycle length, is unaltered in <t>Insm1</t> -/- embryos at E12.5 . (A) At E12.5 there is no difference in the number of OE cells in the cell cycle (those expressing Ki67; P = 0.91) or in S-phase (those having incorporated bromodeoxyuridine (BrdU) 30 minutes after injection; P = 0.29) between Insm1 -/- (grey bars) and Insm1 +/+ littermates (white bars; n = 3 embryo pairs, total of 9 aligned section pairs). (B) The fraction of dividing cells (Ki67+) that are in S-phase (BrdU+), which is indicative of cell cycle length, does not differ between Insm1 -/- mice and their Insm1 +/+ littermates ( P = 0.11). Cell cycle length does not differ among genotypes when considering all progenitors (apical + basal; P = 0.11) or only the basal ones ( P = 0.19). (C) For BrdU incorporation, a larger data set (n = 6 embryo pairs, total of 18 aligned section pairs) also shows no significant difference of cells in S-phase at E12.5 ( P = 0.08). Data are presented as mean values ± standard error of the mean (SEM).
    Insm1 Sequence, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Pacific Biosciences complete minicircle sequences
    Relative positions of specific gRNAs in editing cascades for both strains. The <t>minicircle-encoded</t> and maxicircle-encoded gRNAs were mapped to the 12 mature edited maxicircle transcript sequences. The edited sequences (CO3, Cyb, Murf2 and A6) are in green, the editing domains are in orange and the minicircle-encoded gRNAs are in red. Maxicircle-encoded gRNAs are shown as blue arrows labeled “MC gRNA” above the sequences. The gRNAs are labeled with the gene name and Roman numerals which refer to the location in the functional editing cascade; the low numbers indicate roles early in the cascade and the higher numbers roles later in the cascade. Redundant gRNAs are indicated with a, b, etc. To easily compare the extent of gRNA coverage in the two strains, the editing domains for each gene in both strains are extracted together with the gRNA location information and shown side by side. Apparently missing overlapping gRNAs are indicated by blue circles.
    Complete Minicircle Sequences, supplied by Pacific Biosciences, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    TaKaRa vf1 encoding sequence
    <t>VF1</t> antagonizes the innate immune response to MNV infection. (A) Time course of ISG54 and CXCL10 mRNA expression in RAW264.7 cells infected at an MOI of 0.1 TCID 50 per cell. mRNA levels were quantified by qPCR using an endogenous control gene (Hypoxanthine-guanine phosphoribosyltransferase, HPRT). Expression of the respective mRNAs was then calculated using the ΔΔCt method to compare infected and mock infected cells. Relative fold change was calculated using mock infected samples taken at comparable time points. Normalization was performed following MNV quantification in each sample. Infections were carried out in triplicate with each sample being subsequently analyzed in duplicate by qPCR. The error bars denote standard deviation from the mean. Statistical analysis was performed using an unpaired two tailed t test. (B) Time course of IFN-Beta mRNA and protein production in RAW264.7 cells infected as in (A). mRNA upregulation was calculated as described for (A). Interferon Beta protein was quantified by murine IFN-B specific ELISA at 24 hpi from supernatant samples taken from infected cells. For the ELISA infections were carried out in sextuplate. The error bars denote standard deviation from the mean. (C) MEF cells transfected with an IFN-Beta promoter driven luciferase reporter as well as expression constructs for RIG1, MDA5, MAVS or TBK1 were co-transfected with plasmid DNA expressing VF1 or blank message. Luciferase production was assayed 24 hours post transfection as described in the Materials and Methods . Transfections were carried out in triplicate. The error bars denote standard deviation from the mean. Statistical analysis was performed using an unpaired two tailed t test.
    Vf1 Encoding Sequence, supplied by TaKaRa, used in various techniques. Bioz Stars score: 78/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Biotechnology Information rice pho1 primary sequence
    The Change in Chain Length Distribution of Amylopectin in Rice Endosperm between Various <t>pho1</t> Mutants and Their Wild-Type Parents (T65 or Kinmaze) as Determined by the APTS-Capillary Electrophoresis Method.
    Rice Pho1 Primary Sequence, supplied by Biotechnology Information, used in various techniques. Bioz Stars score: 78/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Sigma-Genosys cgi 58 sequence
    Cryosubstituted, Cryofixed Postembedding Immunoelectron Microscopy with <t>Anti-CGI-58</t> Antiserum in Normal Human Epidermis. A: Anti-CGI-58 antibody deposition labeled with 5 nm gold particles was seen on cytoplasmic vesicles ( arrows ) in the uppermost spinous
    Cgi 58 Sequence, supplied by Sigma-Genosys, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher m pvtrag38 sequences
    <t>PvTRAg38</t> expressed on the CHO-K1 cell surface binds to human erythrocytes. The transfected CHO-K1 cells were incubated with mouse monoclonal antibody DL6 directed against the C terminus of the H. simplex glycoprotein D sequences and then stained with
    M Pvtrag38 Sequences, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Mine Safety Appliances coq6p sequence homologs
    Molecular dynamics of <t>Coq6p</t> homology models. A): Time-evolution of substrate access channel diameter at choke points over MD runs for Coq6p_MODELLER homology model: wild-type (blue trace), Coq6p-G248R (red trace) and Coq6p-L382E (green trace) single mutants, and the Coq6p-G248R-L382E double mutant (purple trace). B): In the wild-type, the channel diameter is large and is constitutively open. C-D): The single mutants display consistently reduced channel diameters which show thermal fluctuations around open and closed states. E): The double mutant displays a channel diameter that is always below the minimum required for substrate binding. In this figure, re face tunnel 1 (purple volume) is oriented directly to the viewer. The pair of residues tracked of the MD calculations as bottlenecks of the channel are represented in sticks.
    Coq6p Sequence Homologs, supplied by Mine Safety Appliances, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore c terminal fndc5 sequence
    Characterization of the <t>FNDC5</t> antibody. Western blot of mouse serum (1, 2), murine M. quadriceps femoris (3, 4), bovine plasma (5, 6), bovine M. longissimus (7), and bovine M. semitendinosus (8) with antibody against C-terminus of FNDC5 (left panel). The specific band of ∼25 kDa is marked by a red arrow. The right panel shows a parallel blot where the antibody was blocked with the recombinant antigen peptide prior to incubation. This antibody was raised against aa 149–178 of human FNDC5 corresponding to aa 143–171 in cattle ( Figure 3 , transcript T1–T1).
    C Terminal Fndc5 Sequence, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Pharmacological inhibition of IKK β inhibits adipocyte differentiation. (A) Oil red O staining of 3T3-L1 cells induced by differentiation medium or medium containing IKKβ inhibitor BMS-345541 at indicated concentrations. (B) Analysis of IKKβ, Smurf2, and adipogenic genes in 3T3-L1 cells treated with control or 10 µM BMS-345541 for 48 h by QPCR ( n = 3–5). (C) 3T3-L1 cells were treated with vehicle control or 10 µM BMS-345541 for 48 h before incubating with vehicle control or 100 nM PS-341 for 4 h. β-catenin was immunoprecipitated with anti–β-catenin antibodies, and then probed with antiubiquitin monoclonal antibodies. The whole-cell lysates were probed with anti–β-catenin antibodies as an internal control. (D) Western blot analysis of nuclear β-catenin levels in 3T3-L1 cells treated with vehicle control or 10 µM BMS-345541 for 48 h. Nuclear proteins were probed with anti-Histone H3 antibodies as an internal control. (E) Oil red O staining of 3T3-L1 cells induced by differentiation medium or medium containing IKKβ inhibitor sodium salicylate at indicated concentrations. Similar results were obtained from at least three independent experiments. All data are mean ± SD. **, P

    Journal: The Journal of Experimental Medicine

    Article Title: IKKβ links vascular inflammation to obesity and atherosclerosis

    doi: 10.1084/jem.20131281

    Figure Lengend Snippet: Pharmacological inhibition of IKK β inhibits adipocyte differentiation. (A) Oil red O staining of 3T3-L1 cells induced by differentiation medium or medium containing IKKβ inhibitor BMS-345541 at indicated concentrations. (B) Analysis of IKKβ, Smurf2, and adipogenic genes in 3T3-L1 cells treated with control or 10 µM BMS-345541 for 48 h by QPCR ( n = 3–5). (C) 3T3-L1 cells were treated with vehicle control or 10 µM BMS-345541 for 48 h before incubating with vehicle control or 100 nM PS-341 for 4 h. β-catenin was immunoprecipitated with anti–β-catenin antibodies, and then probed with antiubiquitin monoclonal antibodies. The whole-cell lysates were probed with anti–β-catenin antibodies as an internal control. (D) Western blot analysis of nuclear β-catenin levels in 3T3-L1 cells treated with vehicle control or 10 µM BMS-345541 for 48 h. Nuclear proteins were probed with anti-Histone H3 antibodies as an internal control. (E) Oil red O staining of 3T3-L1 cells induced by differentiation medium or medium containing IKKβ inhibitor sodium salicylate at indicated concentrations. Similar results were obtained from at least three independent experiments. All data are mean ± SD. **, P

    Article Snippet: Short hairpin RNA (shRNA) plasmids targeting the mouse IKKβ gene sequence were provided in the lentivirus plasmid vector pLKO.1-Puro by Sigma-Aldrich.

    Techniques: Inhibition, Staining, Real-time Polymerase Chain Reaction, Immunoprecipitation, Western Blot

    Chronic treatment of mice with IKKβ inhibitor ameliorates diet-induced obesity. (A and B) 8-wk-old male C57BL/6 mice were fed a WD and treated with vehicle or 10 mg/kg body weight of BMS-345541 by daily oral gavage for 8 wk. Body weight (A) was measured weekly and fat and lean mass (B) were measured at the end of feeding study ( n = 11–12 mice). (C) Representative photographs of subcutaneous (Sub) and epididymal (Epi) WAT. (D) Expression of Smurf2 and other NF-κB target genes in WAT were analyzed by QPCR ( n = 4 mice). (E) Western blot analysis of nuclear β-catenin levels in WAT of control or BMS-345541-treated mice. Nuclear proteins were probed with anti-Histone H3 antibodies an internal control. (F) Oil red O staining of adipose SV cells from control or BMS-345541–treated mice induced by differentiation medium. (G) Adipose SV cells isolated from control or BMS-345541-treated mice were incubated with vehicle or 100 nM PS-341 as indicated for 4 h. β-catenin was immunoprecipitated with anti–β-catenin antibodies and then probed with antiubiquitin monoclonal antibodies. The whole cell lysates were probed with anti–β-catenin antibodies as an internal control. Similar results were obtained from at least three independent experiments. All data are mean ± SD. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: IKKβ links vascular inflammation to obesity and atherosclerosis

    doi: 10.1084/jem.20131281

    Figure Lengend Snippet: Chronic treatment of mice with IKKβ inhibitor ameliorates diet-induced obesity. (A and B) 8-wk-old male C57BL/6 mice were fed a WD and treated with vehicle or 10 mg/kg body weight of BMS-345541 by daily oral gavage for 8 wk. Body weight (A) was measured weekly and fat and lean mass (B) were measured at the end of feeding study ( n = 11–12 mice). (C) Representative photographs of subcutaneous (Sub) and epididymal (Epi) WAT. (D) Expression of Smurf2 and other NF-κB target genes in WAT were analyzed by QPCR ( n = 4 mice). (E) Western blot analysis of nuclear β-catenin levels in WAT of control or BMS-345541-treated mice. Nuclear proteins were probed with anti-Histone H3 antibodies an internal control. (F) Oil red O staining of adipose SV cells from control or BMS-345541–treated mice induced by differentiation medium. (G) Adipose SV cells isolated from control or BMS-345541-treated mice were incubated with vehicle or 100 nM PS-341 as indicated for 4 h. β-catenin was immunoprecipitated with anti–β-catenin antibodies and then probed with antiubiquitin monoclonal antibodies. The whole cell lysates were probed with anti–β-catenin antibodies as an internal control. Similar results were obtained from at least three independent experiments. All data are mean ± SD. *, P

    Article Snippet: Short hairpin RNA (shRNA) plasmids targeting the mouse IKKβ gene sequence were provided in the lentivirus plasmid vector pLKO.1-Puro by Sigma-Aldrich.

    Techniques: Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, Isolation, Incubation, Immunoprecipitation

    Deficiency of SMC IKKβ inhibits diet-induced vascular inflammation and atherosclerosis development in LDLR −/− mice. (A and B) Representative hematoxylin and eosin sections in aorta (A) and BCA (B) of 16-wk-old male IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice fed a WD for 12 wk. Bottom panels in A represent the magnification of the boxed areas in aorta shown in top panels. (C and D) Quantitative analysis of the lesion area in the aortic root (C) and BCA (D) of WD-fed IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice ( n = 11–23 mice). Representative oil red O–stained sections were displayed below the quantification data. (E) Expression of proinflammatory cytokines in aortas of mice fed chow or WD for 12 wk was analyzed by QPCR ( n = 5 mice). (F) Sections of aortic root atherosclerotic lesions were stained with antibodies against mouse TNF, MCP-1, or IL-1β, followed by fluorescein-labeled secondary antibodies (red). The nuclei were stained with DAPI (blue). A representative figure from three mice per group and the similar result is shown. All data are means ± SD. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: IKKβ links vascular inflammation to obesity and atherosclerosis

    doi: 10.1084/jem.20131281

    Figure Lengend Snippet: Deficiency of SMC IKKβ inhibits diet-induced vascular inflammation and atherosclerosis development in LDLR −/− mice. (A and B) Representative hematoxylin and eosin sections in aorta (A) and BCA (B) of 16-wk-old male IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice fed a WD for 12 wk. Bottom panels in A represent the magnification of the boxed areas in aorta shown in top panels. (C and D) Quantitative analysis of the lesion area in the aortic root (C) and BCA (D) of WD-fed IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice ( n = 11–23 mice). Representative oil red O–stained sections were displayed below the quantification data. (E) Expression of proinflammatory cytokines in aortas of mice fed chow or WD for 12 wk was analyzed by QPCR ( n = 5 mice). (F) Sections of aortic root atherosclerotic lesions were stained with antibodies against mouse TNF, MCP-1, or IL-1β, followed by fluorescein-labeled secondary antibodies (red). The nuclei were stained with DAPI (blue). A representative figure from three mice per group and the similar result is shown. All data are means ± SD. *, P

    Article Snippet: Short hairpin RNA (shRNA) plasmids targeting the mouse IKKβ gene sequence were provided in the lentivirus plasmid vector pLKO.1-Puro by Sigma-Aldrich.

    Techniques: Mouse Assay, BIA-KA, Staining, Expressing, Real-time Polymerase Chain Reaction, Labeling

    IKKβ regulates β-catenin ubiquitination and adipocyte differentiation. (A) Expression levels of preadipocyte, mural cell, and mature adipocyte markers in 3T3-L1 cells at 0 and 48 h after addition of differentiation media ( n = 3). (B) Western blot analysis of IKKβ and IKKα levels in 3T3-L1 preadipocytes expressing control shRNA or shRNA against IKKβ (shIKKβ). (C) Oil red O staining of control or shIKKβ 3T3-L1 cells induced by differentiation medium. (D) Western blot analysis of nuclear β-catenin protein levels of control or shIKKβ 3T3-L1 cells. Nuclear proteins were also probed with anti-Histone H3 antibodies as an internal control. (E) QPCR analysis of expression of IKKβ and adipogenic genes in control or shIKKβ 3T3-L1 cells ( n = 3–5). (F) Control or shIKKβ 3T3-L1 cells were treated with vehicle control or 100 nM PS-341 as indicated for 4 h. β-catenin was immunoprecipitated with anti–β-catenin antibodies, and then probed with antiubiquitin monoclonal antibodies. The whole cell lysates were probed with anti–β-catenin antibodies as an internal control. (G) QPCR analysis of Smurf2 expression in control or shIKKβ 3T3-L1 cells ( n = 4). (H) Western blot analysis of Smurf2 protein levels in control or shIKKβ 3T3-L1 cells. (I) Oil red O staining of 3T3-L1 cells transfected with control vector or vector expressing IκBαM induced by differentiation medium. (J) Western blot analysis of Smurf2 protein levels in 3T3-L1 cells expressing control or IκBαM vectors. (K) Control or IκBαM-expressing 3T3-L1 cells were treated with vehicle control or 100 nM PS-341 as indicated for 4 h. β-catenin was immunoprecipitated with anti–β-catenin antibodies and then probed with anti-ubiquitin monoclonal antibodies. The whole-cell lysates were probed with anti–β-catenin antibodies as an internal control. (L) Western blot analysis of nuclear β-catenin levels in control or IκBαM-expressing 3T3-L1 cells. Nuclear proteins were probed with anti-Histone H3 antibodies as an internal control. (M) Western blot analysis of Smurf2 levels in adipose SV cells isolated from WD-fed IKKβ F/F LDLR −/− (F/F) and SM22Cre + IKKβ F/F LDLR −/− (KO) mice. (N) Adipose SV cells were treated with vehicle control or 100 nM PS-341, as indicated, for 4 h. β-catenin was immunoprecipitated with anti–β-catenin antibodies and then probed with anti-ubiquitin monoclonal antibodies. The whole cell lysates were probed with anti-β-catenin antibodies as an internal control. (O) Western blot analysis of nuclear β-catenin levels in SV cells. Nuclear proteins were probed with anti-Histone H3 antibodies as internal control. Similar results were obtained from at least three independent experiments. All data are means ± SD. **, P

    Journal: The Journal of Experimental Medicine

    Article Title: IKKβ links vascular inflammation to obesity and atherosclerosis

    doi: 10.1084/jem.20131281

    Figure Lengend Snippet: IKKβ regulates β-catenin ubiquitination and adipocyte differentiation. (A) Expression levels of preadipocyte, mural cell, and mature adipocyte markers in 3T3-L1 cells at 0 and 48 h after addition of differentiation media ( n = 3). (B) Western blot analysis of IKKβ and IKKα levels in 3T3-L1 preadipocytes expressing control shRNA or shRNA against IKKβ (shIKKβ). (C) Oil red O staining of control or shIKKβ 3T3-L1 cells induced by differentiation medium. (D) Western blot analysis of nuclear β-catenin protein levels of control or shIKKβ 3T3-L1 cells. Nuclear proteins were also probed with anti-Histone H3 antibodies as an internal control. (E) QPCR analysis of expression of IKKβ and adipogenic genes in control or shIKKβ 3T3-L1 cells ( n = 3–5). (F) Control or shIKKβ 3T3-L1 cells were treated with vehicle control or 100 nM PS-341 as indicated for 4 h. β-catenin was immunoprecipitated with anti–β-catenin antibodies, and then probed with antiubiquitin monoclonal antibodies. The whole cell lysates were probed with anti–β-catenin antibodies as an internal control. (G) QPCR analysis of Smurf2 expression in control or shIKKβ 3T3-L1 cells ( n = 4). (H) Western blot analysis of Smurf2 protein levels in control or shIKKβ 3T3-L1 cells. (I) Oil red O staining of 3T3-L1 cells transfected with control vector or vector expressing IκBαM induced by differentiation medium. (J) Western blot analysis of Smurf2 protein levels in 3T3-L1 cells expressing control or IκBαM vectors. (K) Control or IκBαM-expressing 3T3-L1 cells were treated with vehicle control or 100 nM PS-341 as indicated for 4 h. β-catenin was immunoprecipitated with anti–β-catenin antibodies and then probed with anti-ubiquitin monoclonal antibodies. The whole-cell lysates were probed with anti–β-catenin antibodies as an internal control. (L) Western blot analysis of nuclear β-catenin levels in control or IκBαM-expressing 3T3-L1 cells. Nuclear proteins were probed with anti-Histone H3 antibodies as an internal control. (M) Western blot analysis of Smurf2 levels in adipose SV cells isolated from WD-fed IKKβ F/F LDLR −/− (F/F) and SM22Cre + IKKβ F/F LDLR −/− (KO) mice. (N) Adipose SV cells were treated with vehicle control or 100 nM PS-341, as indicated, for 4 h. β-catenin was immunoprecipitated with anti–β-catenin antibodies and then probed with anti-ubiquitin monoclonal antibodies. The whole cell lysates were probed with anti-β-catenin antibodies as an internal control. (O) Western blot analysis of nuclear β-catenin levels in SV cells. Nuclear proteins were probed with anti-Histone H3 antibodies as internal control. Similar results were obtained from at least three independent experiments. All data are means ± SD. **, P

    Article Snippet: Short hairpin RNA (shRNA) plasmids targeting the mouse IKKβ gene sequence were provided in the lentivirus plasmid vector pLKO.1-Puro by Sigma-Aldrich.

    Techniques: Expressing, Western Blot, shRNA, Staining, Real-time Polymerase Chain Reaction, Immunoprecipitation, Transfection, Plasmid Preparation, Isolation, Gene Knockout, Mouse Assay

    Deficiency of IKKβ reduces atherosclerosis in LDLR −/− mice in the absence of obesity and severe hyperlipidemia. (A–F) Body weight (A), lean mass (B), fat mass (C), plasma cholesterol levels (D), triglyceride (E), and cholesterol distribution (F) of 16-wk-old male IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice fed a low-fat AIN76 diet for 12 wk ( n = 6–10 mice). (G and H) Quantitative analysis of the lesion area in the aortic root (G) and BCA (H). n = 8–14 mice. (I) Sections of aortic root atherosclerotic lesions were stained with antibodies against mouse TNF, MCP-1, or IL-1β, followed by fluorescein-labeled secondary antibodies (red). The nuclei were stained with DAPI (blue). A representative figure from three mice per group and the similar result is shown. (J) Plasma cytokine levels of AIN76 diet-fed mice ( n = 6 mice). Results are representative of three independent experiments. All data are mean ± SD. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: IKKβ links vascular inflammation to obesity and atherosclerosis

    doi: 10.1084/jem.20131281

    Figure Lengend Snippet: Deficiency of IKKβ reduces atherosclerosis in LDLR −/− mice in the absence of obesity and severe hyperlipidemia. (A–F) Body weight (A), lean mass (B), fat mass (C), plasma cholesterol levels (D), triglyceride (E), and cholesterol distribution (F) of 16-wk-old male IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice fed a low-fat AIN76 diet for 12 wk ( n = 6–10 mice). (G and H) Quantitative analysis of the lesion area in the aortic root (G) and BCA (H). n = 8–14 mice. (I) Sections of aortic root atherosclerotic lesions were stained with antibodies against mouse TNF, MCP-1, or IL-1β, followed by fluorescein-labeled secondary antibodies (red). The nuclei were stained with DAPI (blue). A representative figure from three mice per group and the similar result is shown. (J) Plasma cytokine levels of AIN76 diet-fed mice ( n = 6 mice). Results are representative of three independent experiments. All data are mean ± SD. *, P

    Article Snippet: Short hairpin RNA (shRNA) plasmids targeting the mouse IKKβ gene sequence were provided in the lentivirus plasmid vector pLKO.1-Puro by Sigma-Aldrich.

    Techniques: Mouse Assay, BIA-KA, Staining, Labeling

    IKKβ-deficient mice are resistant to diet-induced obesity. (A–D) Body weight (A), lean mass (B), fat mass (C), and fat percentage (D) of 16-wk-old male IKKβ F/F LDLR −/− (F/F) and SM22Cre + IKKβ F/F LDLR −/− (KO) mice fed a normal chow diet (Chow) or WD for 12 wk ( n = 15–20 mice). (E–G) Food intake (E), oxygen consumption (F), and mean oxygen consumption (G) were monitored in WD-fed IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice ( n = 5 mice). Oxygen consumption data were normalized by lean body mass and mean from 4-d measurements. (H) WAT (epididymal, subcutaneous, perirenal, and omental WAT) and BAT weight measured in WD-fed IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice ( n = 7–8 mice). (I and J) The expression of indicated genes in BAT (I) and WAT (J) was analyzed by QPCR ( n = 4–8 mice). (K) Plasma cytokine levels in chow or WD-fed IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice ( n = 5–7 mice). Results are representative of three independent experiments. All data are means ± SD. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: IKKβ links vascular inflammation to obesity and atherosclerosis

    doi: 10.1084/jem.20131281

    Figure Lengend Snippet: IKKβ-deficient mice are resistant to diet-induced obesity. (A–D) Body weight (A), lean mass (B), fat mass (C), and fat percentage (D) of 16-wk-old male IKKβ F/F LDLR −/− (F/F) and SM22Cre + IKKβ F/F LDLR −/− (KO) mice fed a normal chow diet (Chow) or WD for 12 wk ( n = 15–20 mice). (E–G) Food intake (E), oxygen consumption (F), and mean oxygen consumption (G) were monitored in WD-fed IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice ( n = 5 mice). Oxygen consumption data were normalized by lean body mass and mean from 4-d measurements. (H) WAT (epididymal, subcutaneous, perirenal, and omental WAT) and BAT weight measured in WD-fed IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice ( n = 7–8 mice). (I and J) The expression of indicated genes in BAT (I) and WAT (J) was analyzed by QPCR ( n = 4–8 mice). (K) Plasma cytokine levels in chow or WD-fed IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice ( n = 5–7 mice). Results are representative of three independent experiments. All data are means ± SD. *, P

    Article Snippet: Short hairpin RNA (shRNA) plasmids targeting the mouse IKKβ gene sequence were provided in the lentivirus plasmid vector pLKO.1-Puro by Sigma-Aldrich.

    Techniques: Mouse Assay, Gene Knockout, Expressing, Real-time Polymerase Chain Reaction

    Deficiency of IKKβ protects mice from obesity-associated metabolic disorders . (A–C) Fasting plasma glucose and insulin levels ( n = 10–15 mice; A), GTT (B), and the area under the curve (AUC) of GTT (C) in 16-wk-old male IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice fed a WD for 12 wk ( n = 10–15 mice). (D) Macrophage infiltration in WAT determined by F4/80 staining. (E and F) Representative appearance (E) and hematoxylin and eosin (top) and oil red O–stained (bottom) sections (F) of livers. (G–I) Hepatic cholesterol and triglyceride levels (G), plasma cholesterol and triglyceride levels (H), and plasma cholesterol distribution (I) of WD-fed IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice ( n = 6–10 mice). (J) Hepatic gene expression was analyzed by QPCR ( n = 4–8 mice). Results are representative of three independent experiments. All data are means ± SD. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: IKKβ links vascular inflammation to obesity and atherosclerosis

    doi: 10.1084/jem.20131281

    Figure Lengend Snippet: Deficiency of IKKβ protects mice from obesity-associated metabolic disorders . (A–C) Fasting plasma glucose and insulin levels ( n = 10–15 mice; A), GTT (B), and the area under the curve (AUC) of GTT (C) in 16-wk-old male IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice fed a WD for 12 wk ( n = 10–15 mice). (D) Macrophage infiltration in WAT determined by F4/80 staining. (E and F) Representative appearance (E) and hematoxylin and eosin (top) and oil red O–stained (bottom) sections (F) of livers. (G–I) Hepatic cholesterol and triglyceride levels (G), plasma cholesterol and triglyceride levels (H), and plasma cholesterol distribution (I) of WD-fed IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice ( n = 6–10 mice). (J) Hepatic gene expression was analyzed by QPCR ( n = 4–8 mice). Results are representative of three independent experiments. All data are means ± SD. *, P

    Article Snippet: Short hairpin RNA (shRNA) plasmids targeting the mouse IKKβ gene sequence were provided in the lentivirus plasmid vector pLKO.1-Puro by Sigma-Aldrich.

    Techniques: Mouse Assay, Staining, Expressing, Real-time Polymerase Chain Reaction

    Generation of LDLR −/− mice with SMC-specific IKKβ deficiency. (A) Western blot analysis of IKKβ and IKKα expression in aorta, liver, skeletal muscle (s. muscle), and intestine of IKKβ F/F LDLR −/− (F/F) and SM22Cre + IKKβ F/F LDLR −/− (KO) mice. (B) SMCs isolated from aortas of IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice were stimulated with TNF (20 ng/ml) or vehicle for 30 min. Cells stained with anti-p65 primary antibodies, followed by fluorescein-labeled secondary antibodies (green). The nuclei were visualized with DAPI (blue). Bars, 20 µm. (C) SMCs were stimulated with TNF (20 ng/ml) or vehicle for 30 min. Nuclear proteins were extracted and NF-κB binding activity was determined by electrophoretic mobility shift assay. (D) SMCs were treated with LPS (5 µg/ml) or vehicle control for 3 h. Expression of proinflammatory cytokines was analyzed by QPCR ( n = 5). Similar results were obtained from at least three independent experiments. All data are means ± SD. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: IKKβ links vascular inflammation to obesity and atherosclerosis

    doi: 10.1084/jem.20131281

    Figure Lengend Snippet: Generation of LDLR −/− mice with SMC-specific IKKβ deficiency. (A) Western blot analysis of IKKβ and IKKα expression in aorta, liver, skeletal muscle (s. muscle), and intestine of IKKβ F/F LDLR −/− (F/F) and SM22Cre + IKKβ F/F LDLR −/− (KO) mice. (B) SMCs isolated from aortas of IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice were stimulated with TNF (20 ng/ml) or vehicle for 30 min. Cells stained with anti-p65 primary antibodies, followed by fluorescein-labeled secondary antibodies (green). The nuclei were visualized with DAPI (blue). Bars, 20 µm. (C) SMCs were stimulated with TNF (20 ng/ml) or vehicle for 30 min. Nuclear proteins were extracted and NF-κB binding activity was determined by electrophoretic mobility shift assay. (D) SMCs were treated with LPS (5 µg/ml) or vehicle control for 3 h. Expression of proinflammatory cytokines was analyzed by QPCR ( n = 5). Similar results were obtained from at least three independent experiments. All data are means ± SD. *, P

    Article Snippet: Short hairpin RNA (shRNA) plasmids targeting the mouse IKKβ gene sequence were provided in the lentivirus plasmid vector pLKO.1-Puro by Sigma-Aldrich.

    Techniques: Mouse Assay, Western Blot, Expressing, Gene Knockout, Isolation, Staining, Labeling, Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay, Real-time Polymerase Chain Reaction

    SM22Cre-mediated IKKβ ablation blocks white adipose differentiation. (A) Hematoxylin and eosin staining of transverse sections of subcutaneous WAT from WD-fed IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice. The adipocyte numbers were calculated based on per mm epidermal length ( n = 5 mice). (B) Schematic of the SM22Cre + Rosa26 EGFP mouse model. (C) Quantitation of GFP + cells in cultures of adipose SV cells isolated from Rosa26 EGFP and SM22Cre + Rosa26 EGFP mice by flow cytometry. The percentage of GFP + cells are as indicated in the flow profiles. (D) PCR analysis of genomic DNA from adipose SV cells of IKKβ F/F LDLR −/− (F/F) and SM22Cre + IKKβ F/F LDLR −/− (KO) mice (top). Expression levels of IKKβ, inflammatory genes, and leptin in adipose SV cells were analyzed by QPCR ( n = 3 mice) (bottom). (E) Oil red O staining of adipose SV cells of IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice induced by differentiation medium. The nuclei were stained with hematoxylin (blue) in the bottom panel. (F) SV cells and mature adipocytes were isolated from WAT of WD-fed IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice. The cell population was displayed as cell number per gram of WAT ( n = 5 mice). (G) Expression of adipogenic genes and adipocyte precursor cell markers in adipose SV cells was measured by QPCR ( n = 5–7 mice). (H) Sections of subcutaneous WAT were stained with antibodies against adipocyte progenitor markers PDGFRβ and preadipocyte marker Pref-1, followed by Alexa Fluor 488 (green)– or Alexa Fluor 594 (red)–labeled secondary antibodies. A representative figure from three mice per group and the similar result is shown. (I) Adipose SV cells were examined for expression of adipocyte progenitor cell makers, PDGFRβ, and NG2, and endothelial cell marker CD31 with flow cytometry. CD31 + , NG2 + , and CD31 − NG2 − cells are highlighted with red, green, and black, respectively. The percentages of CD31 − PDGFRβ + NG2 + cells are as indicated in the flow profiles (P

    Journal: The Journal of Experimental Medicine

    Article Title: IKKβ links vascular inflammation to obesity and atherosclerosis

    doi: 10.1084/jem.20131281

    Figure Lengend Snippet: SM22Cre-mediated IKKβ ablation blocks white adipose differentiation. (A) Hematoxylin and eosin staining of transverse sections of subcutaneous WAT from WD-fed IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice. The adipocyte numbers were calculated based on per mm epidermal length ( n = 5 mice). (B) Schematic of the SM22Cre + Rosa26 EGFP mouse model. (C) Quantitation of GFP + cells in cultures of adipose SV cells isolated from Rosa26 EGFP and SM22Cre + Rosa26 EGFP mice by flow cytometry. The percentage of GFP + cells are as indicated in the flow profiles. (D) PCR analysis of genomic DNA from adipose SV cells of IKKβ F/F LDLR −/− (F/F) and SM22Cre + IKKβ F/F LDLR −/− (KO) mice (top). Expression levels of IKKβ, inflammatory genes, and leptin in adipose SV cells were analyzed by QPCR ( n = 3 mice) (bottom). (E) Oil red O staining of adipose SV cells of IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice induced by differentiation medium. The nuclei were stained with hematoxylin (blue) in the bottom panel. (F) SV cells and mature adipocytes were isolated from WAT of WD-fed IKKβ F/F LDLR −/− and SM22Cre + IKKβ F/F LDLR −/− mice. The cell population was displayed as cell number per gram of WAT ( n = 5 mice). (G) Expression of adipogenic genes and adipocyte precursor cell markers in adipose SV cells was measured by QPCR ( n = 5–7 mice). (H) Sections of subcutaneous WAT were stained with antibodies against adipocyte progenitor markers PDGFRβ and preadipocyte marker Pref-1, followed by Alexa Fluor 488 (green)– or Alexa Fluor 594 (red)–labeled secondary antibodies. A representative figure from three mice per group and the similar result is shown. (I) Adipose SV cells were examined for expression of adipocyte progenitor cell makers, PDGFRβ, and NG2, and endothelial cell marker CD31 with flow cytometry. CD31 + , NG2 + , and CD31 − NG2 − cells are highlighted with red, green, and black, respectively. The percentages of CD31 − PDGFRβ + NG2 + cells are as indicated in the flow profiles (P

    Article Snippet: Short hairpin RNA (shRNA) plasmids targeting the mouse IKKβ gene sequence were provided in the lentivirus plasmid vector pLKO.1-Puro by Sigma-Aldrich.

    Techniques: Staining, Mouse Assay, Quantitation Assay, Isolation, Flow Cytometry, Cytometry, Polymerase Chain Reaction, Gene Knockout, Expressing, Real-time Polymerase Chain Reaction, Marker, Labeling

    Immunoblots showing co-IP of ZO-1 and Cx47 from various brain regions and from Cx47-transfected HeLa cells. (A) Homogenates of brain regions were taken for IP with anti-ZO-1 antibody and immunoblots of precipitates were probed with anti-Cx47 antibody.

    Journal:

    Article Title: CONNEXIN47, CONNEXIN29 AND CONNEXIN32 CO-EXPRESSION IN OLIGODENDROCYTES AND Cx47 ASSOCIATION WITH ZONULA OCCLUDENS-1 (ZO-1) IN MOUSE BRAIN

    doi: 10.1016/j.neuroscience.2004.03.063

    Figure Lengend Snippet: Immunoblots showing co-IP of ZO-1 and Cx47 from various brain regions and from Cx47-transfected HeLa cells. (A) Homogenates of brain regions were taken for IP with anti-ZO-1 antibody and immunoblots of precipitates were probed with anti-Cx47 antibody.

    Article Snippet: Oligonucleotide primers, materials for amplifying mouse Cx47 coding sequence, and transfection reagents were purchased from Gibco BRL Life Technologies (Burlington, Ontario, Canada).

    Techniques: Western Blot, Co-Immunoprecipitation Assay, Transfection

    Pull-down assays showing Cx47 interaction with the second PDZ domain of ZO-1. (A) Immunoblots of lysates from empty vector-transfected HeLa cells (lane 1), Cx47-transfected HeLa cells (lane 2) and homogenate from brain tissue (lane 3) show negative and

    Journal:

    Article Title: CONNEXIN47, CONNEXIN29 AND CONNEXIN32 CO-EXPRESSION IN OLIGODENDROCYTES AND Cx47 ASSOCIATION WITH ZONULA OCCLUDENS-1 (ZO-1) IN MOUSE BRAIN

    doi: 10.1016/j.neuroscience.2004.03.063

    Figure Lengend Snippet: Pull-down assays showing Cx47 interaction with the second PDZ domain of ZO-1. (A) Immunoblots of lysates from empty vector-transfected HeLa cells (lane 1), Cx47-transfected HeLa cells (lane 2) and homogenate from brain tissue (lane 3) show negative and

    Article Snippet: Oligonucleotide primers, materials for amplifying mouse Cx47 coding sequence, and transfection reagents were purchased from Gibco BRL Life Technologies (Burlington, Ontario, Canada).

    Techniques: Western Blot, Plasmid Preparation, Transfection

    FRIL labeling of Cx47 in oligodendrocytes in adult rat spinal cord. (A) In a small area of oligodendrocyte soma, Cx47-immunoreactivity (12 nm gold) is seen in each of five gap junctions, which range from about 100 to more than 500 connexons. (B) At higher

    Journal:

    Article Title: CONNEXIN47, CONNEXIN29 AND CONNEXIN32 CO-EXPRESSION IN OLIGODENDROCYTES AND Cx47 ASSOCIATION WITH ZONULA OCCLUDENS-1 (ZO-1) IN MOUSE BRAIN

    doi: 10.1016/j.neuroscience.2004.03.063

    Figure Lengend Snippet: FRIL labeling of Cx47 in oligodendrocytes in adult rat spinal cord. (A) In a small area of oligodendrocyte soma, Cx47-immunoreactivity (12 nm gold) is seen in each of five gap junctions, which range from about 100 to more than 500 connexons. (B) At higher

    Article Snippet: Oligonucleotide primers, materials for amplifying mouse Cx47 coding sequence, and transfection reagents were purchased from Gibco BRL Life Technologies (Burlington, Ontario, Canada).

    Techniques: Labeling

    Laser scanning confocal immunofluorescence of Cx47-immunopositive puncta on CNPase-labeled oligodendrocyte somata, and their co-localization with Cx32 and Cx43. (A) Z-stack of 34 scans showing the same oligodendrocyte rotated through 135° at 45°

    Journal:

    Article Title: CONNEXIN47, CONNEXIN29 AND CONNEXIN32 CO-EXPRESSION IN OLIGODENDROCYTES AND Cx47 ASSOCIATION WITH ZONULA OCCLUDENS-1 (ZO-1) IN MOUSE BRAIN

    doi: 10.1016/j.neuroscience.2004.03.063

    Figure Lengend Snippet: Laser scanning confocal immunofluorescence of Cx47-immunopositive puncta on CNPase-labeled oligodendrocyte somata, and their co-localization with Cx32 and Cx43. (A) Z-stack of 34 scans showing the same oligodendrocyte rotated through 135° at 45°

    Article Snippet: Oligonucleotide primers, materials for amplifying mouse Cx47 coding sequence, and transfection reagents were purchased from Gibco BRL Life Technologies (Burlington, Ontario, Canada).

    Techniques: Immunofluorescence, Labeling

    Immunofluorescence co-localization relationships of ZO-1 with CNPase, Cx47 and Cx32. (A) Confocal double immunofluorescence of a CNPase-positive oligodendrocyte somata (A1, arrow) displaying punctate labeling for ZO-1 (A2, arrow) as shown in overlay (A3).

    Journal:

    Article Title: CONNEXIN47, CONNEXIN29 AND CONNEXIN32 CO-EXPRESSION IN OLIGODENDROCYTES AND Cx47 ASSOCIATION WITH ZONULA OCCLUDENS-1 (ZO-1) IN MOUSE BRAIN

    doi: 10.1016/j.neuroscience.2004.03.063

    Figure Lengend Snippet: Immunofluorescence co-localization relationships of ZO-1 with CNPase, Cx47 and Cx32. (A) Confocal double immunofluorescence of a CNPase-positive oligodendrocyte somata (A1, arrow) displaying punctate labeling for ZO-1 (A2, arrow) as shown in overlay (A3).

    Article Snippet: Oligonucleotide primers, materials for amplifying mouse Cx47 coding sequence, and transfection reagents were purchased from Gibco BRL Life Technologies (Burlington, Ontario, Canada).

    Techniques: Immunofluorescence, Labeling

    Regional expression of Cx47 protein in adult mouse brain. Immunoblots show a migration profile of Cx47 in brain (lanes 1–7) comparable to that observed in Cx47-transfected HeLa cells (lane 9), and an absence of Cx47 in control empty vector-transfected

    Journal:

    Article Title: CONNEXIN47, CONNEXIN29 AND CONNEXIN32 CO-EXPRESSION IN OLIGODENDROCYTES AND Cx47 ASSOCIATION WITH ZONULA OCCLUDENS-1 (ZO-1) IN MOUSE BRAIN

    doi: 10.1016/j.neuroscience.2004.03.063

    Figure Lengend Snippet: Regional expression of Cx47 protein in adult mouse brain. Immunoblots show a migration profile of Cx47 in brain (lanes 1–7) comparable to that observed in Cx47-transfected HeLa cells (lane 9), and an absence of Cx47 in control empty vector-transfected

    Article Snippet: Oligonucleotide primers, materials for amplifying mouse Cx47 coding sequence, and transfection reagents were purchased from Gibco BRL Life Technologies (Burlington, Ontario, Canada).

    Techniques: Expressing, Western Blot, Migration, Transfection, Plasmid Preparation

    Immunofluorescence labeling of Cx47

    Journal:

    Article Title: CONNEXIN47, CONNEXIN29 AND CONNEXIN32 CO-EXPRESSION IN OLIGODENDROCYTES AND Cx47 ASSOCIATION WITH ZONULA OCCLUDENS-1 (ZO-1) IN MOUSE BRAIN

    doi: 10.1016/j.neuroscience.2004.03.063

    Figure Lengend Snippet: Immunofluorescence labeling of Cx47

    Article Snippet: Oligonucleotide primers, materials for amplifying mouse Cx47 coding sequence, and transfection reagents were purchased from Gibco BRL Life Technologies (Burlington, Ontario, Canada).

    Techniques: Immunofluorescence, Labeling

    Western blots showing the subcellular distribution of Cx47, Cx32 and Cx29 in adult mouse brain. Following subcellular fractionation, the same immunoblot loaded with equal levels of protein from whole brain homogenate (lane 1), soluble (lane 2), microsomal

    Journal:

    Article Title: CONNEXIN47, CONNEXIN29 AND CONNEXIN32 CO-EXPRESSION IN OLIGODENDROCYTES AND Cx47 ASSOCIATION WITH ZONULA OCCLUDENS-1 (ZO-1) IN MOUSE BRAIN

    doi: 10.1016/j.neuroscience.2004.03.063

    Figure Lengend Snippet: Western blots showing the subcellular distribution of Cx47, Cx32 and Cx29 in adult mouse brain. Following subcellular fractionation, the same immunoblot loaded with equal levels of protein from whole brain homogenate (lane 1), soluble (lane 2), microsomal

    Article Snippet: Oligonucleotide primers, materials for amplifying mouse Cx47 coding sequence, and transfection reagents were purchased from Gibco BRL Life Technologies (Burlington, Ontario, Canada).

    Techniques: Western Blot, Fractionation

    Low magnification immunofluorescence micrographs showing the distribution of Cx47-immunopositive cells in various regions of adult mouse CNS. Labeling for Cx47 is seen associated with oligodendrocyte cell bodies in all layers of the cerebral cortex (A),

    Journal:

    Article Title: CONNEXIN47, CONNEXIN29 AND CONNEXIN32 CO-EXPRESSION IN OLIGODENDROCYTES AND Cx47 ASSOCIATION WITH ZONULA OCCLUDENS-1 (ZO-1) IN MOUSE BRAIN

    doi: 10.1016/j.neuroscience.2004.03.063

    Figure Lengend Snippet: Low magnification immunofluorescence micrographs showing the distribution of Cx47-immunopositive cells in various regions of adult mouse CNS. Labeling for Cx47 is seen associated with oligodendrocyte cell bodies in all layers of the cerebral cortex (A),

    Article Snippet: Oligonucleotide primers, materials for amplifying mouse Cx47 coding sequence, and transfection reagents were purchased from Gibco BRL Life Technologies (Burlington, Ontario, Canada).

    Techniques: Immunofluorescence, Labeling

    Double immunofluorescence micrographs showing relationships of cells labeled for CNPase, Cx32 and Cx47 in adult mouse brain. (A–D) The same fields in the hippocampus (A, B) and hypothalamus (C, D) showing correspondence of cells (arrows) labeled

    Journal:

    Article Title: CONNEXIN47, CONNEXIN29 AND CONNEXIN32 CO-EXPRESSION IN OLIGODENDROCYTES AND Cx47 ASSOCIATION WITH ZONULA OCCLUDENS-1 (ZO-1) IN MOUSE BRAIN

    doi: 10.1016/j.neuroscience.2004.03.063

    Figure Lengend Snippet: Double immunofluorescence micrographs showing relationships of cells labeled for CNPase, Cx32 and Cx47 in adult mouse brain. (A–D) The same fields in the hippocampus (A, B) and hypothalamus (C, D) showing correspondence of cells (arrows) labeled

    Article Snippet: Oligonucleotide primers, materials for amplifying mouse Cx47 coding sequence, and transfection reagents were purchased from Gibco BRL Life Technologies (Burlington, Ontario, Canada).

    Techniques: Immunofluorescence, Labeling

    Immunofluorescence labeling of Cx47 and ZO-1 in cultured HeLa cells. (A) Double immunofluorescence of the same field showing punctate labeling of Cx47 (A1) and ZO-1 (A2) at points of cell-to-cell contact (arrows) in HeLa cells stably transfected with

    Journal:

    Article Title: CONNEXIN47, CONNEXIN29 AND CONNEXIN32 CO-EXPRESSION IN OLIGODENDROCYTES AND Cx47 ASSOCIATION WITH ZONULA OCCLUDENS-1 (ZO-1) IN MOUSE BRAIN

    doi: 10.1016/j.neuroscience.2004.03.063

    Figure Lengend Snippet: Immunofluorescence labeling of Cx47 and ZO-1 in cultured HeLa cells. (A) Double immunofluorescence of the same field showing punctate labeling of Cx47 (A1) and ZO-1 (A2) at points of cell-to-cell contact (arrows) in HeLa cells stably transfected with

    Article Snippet: Oligonucleotide primers, materials for amplifying mouse Cx47 coding sequence, and transfection reagents were purchased from Gibco BRL Life Technologies (Burlington, Ontario, Canada).

    Techniques: Immunofluorescence, Labeling, Cell Culture, Stable Transfection, Transfection

    TGF-β 1 associates with areas of decreased clusterin expression in fibrotic lung and down-regulates fibroblast clusterin mRNA and protein expression in vitro . Serial sections prepared from IPF lung (n = 3) were stained immunohistochemically to localize TGF-β 1 staining (( A ), red/brown, nuclei blue) and clusterin (( B ), red/brown, nuclei blue) in fibroblastic foci. ( A,B ) Representative images of immunohistochemical staining suggest that TGF-β 1 localizes to ECM, fibroblasts and macrophages, whilst staining for clusterin is weak or undetectable. ( C ) In vitro analysis of clusterin mRNA levels in TGF-β 1 stimulated (40 pM) human lung fibroblasts was performed via qRT-PCR and shows a time-dependent down-regulation of clusterin expression that was maximal at 24–48 h (n = 3) in response to TGF-β 1 . Clusterin protein levels were also down-regulated in response to TGF-β 1 (40 pM) compared to control at 24 h and 48 h as demonstrated by western blotting at 24 h ( D ) and immunofluorescent staining at 48 h (E, red, nuclei - blue). ( F ) Semi-quantitative analysis of fluorescent signal of panel E: clusterin signal (pixel intensity) was normalized to cell numbers per visual field and compared to control (n = 6). Full-length western blots are presented in Supplementary Figure e4 . Different cell populations/structures are indicated by arrows; f - fibroblast-like cell, m - macrophage, he - hyperplastic epithelial cell, ecm – extracellular matrix. * P

    Journal: Scientific Reports

    Article Title: Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis

    doi: 10.1038/s41598-018-20316-1

    Figure Lengend Snippet: TGF-β 1 associates with areas of decreased clusterin expression in fibrotic lung and down-regulates fibroblast clusterin mRNA and protein expression in vitro . Serial sections prepared from IPF lung (n = 3) were stained immunohistochemically to localize TGF-β 1 staining (( A ), red/brown, nuclei blue) and clusterin (( B ), red/brown, nuclei blue) in fibroblastic foci. ( A,B ) Representative images of immunohistochemical staining suggest that TGF-β 1 localizes to ECM, fibroblasts and macrophages, whilst staining for clusterin is weak or undetectable. ( C ) In vitro analysis of clusterin mRNA levels in TGF-β 1 stimulated (40 pM) human lung fibroblasts was performed via qRT-PCR and shows a time-dependent down-regulation of clusterin expression that was maximal at 24–48 h (n = 3) in response to TGF-β 1 . Clusterin protein levels were also down-regulated in response to TGF-β 1 (40 pM) compared to control at 24 h and 48 h as demonstrated by western blotting at 24 h ( D ) and immunofluorescent staining at 48 h (E, red, nuclei - blue). ( F ) Semi-quantitative analysis of fluorescent signal of panel E: clusterin signal (pixel intensity) was normalized to cell numbers per visual field and compared to control (n = 6). Full-length western blots are presented in Supplementary Figure e4 . Different cell populations/structures are indicated by arrows; f - fibroblast-like cell, m - macrophage, he - hyperplastic epithelial cell, ecm – extracellular matrix. * P

    Article Snippet: Lentiviral plasmids with short hairpin sequences targeting clusterin were obtained from GE Healthcare UK.

    Techniques: Expressing, In Vitro, Staining, Immunohistochemistry, Quantitative RT-PCR, Western Blot

    Localization of clusterin in IPF lung. Immunohistochemical staining for clusterin was performed on formalin-fixed, paraffin embedded 3 μm sections of IPF lung tissue (n = 3). Clusterin staining (clu, A, C-G, I brown/red, nuclei - blue) and staining for elastin (H, J, EvG, grey/black) in representative tissue sections. Clusterin is undetectable in αSMA positive myofibroblasts ( A,B ), forming and in cells overlying fibroblastic foci ( A,C ), compared to strong staining of fibroblast-like cells in morphologically normal non-fibrotic areas ( D ). Clusterin was observed sporadically in bronchial epithelial cells but more frequently than in controls ( E ). Similar to control lung, clusterin colocalized with elastin ( G,H ) and was undetectable in macrophages, smooth muscle and endothelial cells ( F,G ). Clusterin also colocalized with amorphous elastin aggregates in dense fibrotic regions ( I,J ). Different cell populations/structures are indicated by arrows; f - fibroblast-like cell, m - macrophage, he - hyperplastic epithelial cell, be - bronchial epithelial cell, en – endothelial cell, smc – smooth muscle cell, ef- elastic fibers. Scale bar represents 25 µm ( A–J ).

    Journal: Scientific Reports

    Article Title: Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis

    doi: 10.1038/s41598-018-20316-1

    Figure Lengend Snippet: Localization of clusterin in IPF lung. Immunohistochemical staining for clusterin was performed on formalin-fixed, paraffin embedded 3 μm sections of IPF lung tissue (n = 3). Clusterin staining (clu, A, C-G, I brown/red, nuclei - blue) and staining for elastin (H, J, EvG, grey/black) in representative tissue sections. Clusterin is undetectable in αSMA positive myofibroblasts ( A,B ), forming and in cells overlying fibroblastic foci ( A,C ), compared to strong staining of fibroblast-like cells in morphologically normal non-fibrotic areas ( D ). Clusterin was observed sporadically in bronchial epithelial cells but more frequently than in controls ( E ). Similar to control lung, clusterin colocalized with elastin ( G,H ) and was undetectable in macrophages, smooth muscle and endothelial cells ( F,G ). Clusterin also colocalized with amorphous elastin aggregates in dense fibrotic regions ( I,J ). Different cell populations/structures are indicated by arrows; f - fibroblast-like cell, m - macrophage, he - hyperplastic epithelial cell, be - bronchial epithelial cell, en – endothelial cell, smc – smooth muscle cell, ef- elastic fibers. Scale bar represents 25 µm ( A–J ).

    Article Snippet: Lentiviral plasmids with short hairpin sequences targeting clusterin were obtained from GE Healthcare UK.

    Techniques: Immunohistochemistry, Staining, Formalin-fixed Paraffin-Embedded

    Effect of clusterin deficiency on apoptosis. Lung fibroblasts were transduced with clusterin shRNA (shCLU, open circles) and mock shRNA vectors (grey circles) or remained untransfected (black circles). Lung fibroblasts were seeded and treated with FasL (3 nM – 6 nM) and/or exogenous clusterin (CLU, 125 nM) for 19 h or remained untreated. Apoptotic cells (Annexin V+ and Annexin V+/ DAPI+ cells) were assessed via FACS analysis post staining of apoptotic cells with annexin V – Alexa647 and DAPI (mean ± SEM, n = 5). ( A ) shRNA-induced clusterin deficiency sensitized fibroblasts to basal and FasL-induced apoptosis, and this could be overcome by addition of exogenous clusterin ( B ). ( C ) Basal apoptosis in representative control (9.37 ± 0.63%) and fibrotic (11.6 ± 0.69%) lung fibroblast isolates was not significantly different. Fibrotic lung fibroblasts were more resistant to FasL-induced apoptosis compared with controls ( C ) and exogenous clusterin tends to reduce basal and FasL-induced apoptotic levels further ( D ). Data representative of at least two individual experiments with fibroblasts derived from 1 donor per group. *P

    Journal: Scientific Reports

    Article Title: Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis

    doi: 10.1038/s41598-018-20316-1

    Figure Lengend Snippet: Effect of clusterin deficiency on apoptosis. Lung fibroblasts were transduced with clusterin shRNA (shCLU, open circles) and mock shRNA vectors (grey circles) or remained untransfected (black circles). Lung fibroblasts were seeded and treated with FasL (3 nM – 6 nM) and/or exogenous clusterin (CLU, 125 nM) for 19 h or remained untreated. Apoptotic cells (Annexin V+ and Annexin V+/ DAPI+ cells) were assessed via FACS analysis post staining of apoptotic cells with annexin V – Alexa647 and DAPI (mean ± SEM, n = 5). ( A ) shRNA-induced clusterin deficiency sensitized fibroblasts to basal and FasL-induced apoptosis, and this could be overcome by addition of exogenous clusterin ( B ). ( C ) Basal apoptosis in representative control (9.37 ± 0.63%) and fibrotic (11.6 ± 0.69%) lung fibroblast isolates was not significantly different. Fibrotic lung fibroblasts were more resistant to FasL-induced apoptosis compared with controls ( C ) and exogenous clusterin tends to reduce basal and FasL-induced apoptotic levels further ( D ). Data representative of at least two individual experiments with fibroblasts derived from 1 donor per group. *P

    Article Snippet: Lentiviral plasmids with short hairpin sequences targeting clusterin were obtained from GE Healthcare UK.

    Techniques: Transduction, shRNA, FACS, Staining, Derivative Assay

    Clusterin gene expression and protein levels are decreased in fibrotic compared with control lung fibroblasts. Fibroblasts isolated from human control and fibrotic lung were grown in monolayer culture and clusterin mRNA and protein levels were detected via microarray, proteome profiler and immunofluoresecence analysis. ( A ) Microarray analysis of mRNA shows decreased clusterin gene expression in fibroblasts derived from fibrotic lungs (open circles; n = 5 IPF and 7 SSc) compared with controls (closed circles; n = 6). Proteome profiler array analysis ( B , clusterin - CLU) and immunofluorescence staining of control and fibrotic lung fibroblasts ( C,D ) confirms low clusterin protein expression in fibrotic compared with control fibroblasts in vitr o. ( E ) Semi-quantitative analysis of clusterin staining ( C,D ), clusterin signal (pixel intensity) normalized to cell numbers per visual field (n = 6). Data is representative of three individual experiments. * P

    Journal: Scientific Reports

    Article Title: Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis

    doi: 10.1038/s41598-018-20316-1

    Figure Lengend Snippet: Clusterin gene expression and protein levels are decreased in fibrotic compared with control lung fibroblasts. Fibroblasts isolated from human control and fibrotic lung were grown in monolayer culture and clusterin mRNA and protein levels were detected via microarray, proteome profiler and immunofluoresecence analysis. ( A ) Microarray analysis of mRNA shows decreased clusterin gene expression in fibroblasts derived from fibrotic lungs (open circles; n = 5 IPF and 7 SSc) compared with controls (closed circles; n = 6). Proteome profiler array analysis ( B , clusterin - CLU) and immunofluorescence staining of control and fibrotic lung fibroblasts ( C,D ) confirms low clusterin protein expression in fibrotic compared with control fibroblasts in vitr o. ( E ) Semi-quantitative analysis of clusterin staining ( C,D ), clusterin signal (pixel intensity) normalized to cell numbers per visual field (n = 6). Data is representative of three individual experiments. * P

    Article Snippet: Lentiviral plasmids with short hairpin sequences targeting clusterin were obtained from GE Healthcare UK.

    Techniques: Expressing, Isolation, Microarray, Derivative Assay, Immunofluorescence, Staining

    Effect of clusterin deficiency on TGF-β 1 -induced myofibroblast differentiation and collagen deposition. Lung fibroblasts were transduced with clusterin shRNA (shCLU, open bars) and mock shRNA vectors (grey bars) or remained untransfected (black bars). αSMA mRNA was assessed via qRT-PCR ( A ) and clusterin and αSMA protein levels detected by western blotting ( B,C quantification). 48 h following TGF-β 1 stimulation (40 pM) αSMA mRNA and protein were increased. Basal and increased levels of αSMA mRNA and protein, however, did not vary between clusterin deficient, mock-transduced and control fibroblasts. Collagen mRNA ( D ) and deposition ( E , F quantification) assessed by qRT-PCR and immunofluorescence staining were significantly increased in response to TGF-β 1 . Although, basal and TGF-β 1 induced changes in collagen levels varied between clusterin deficient, mock and control fibroblasts, the overall fold-increase in collagen mRNA and deposition levels did not significantly change between clusterin deficient fibroblasts and control/mock fibroblasts. Data generated in A-F is representative of two individual experiments with fibroblasts derived from 2 donors. Full-length western blots are presented in Supplementary Figure e5 . Scale bar in E represents 10 µm. ** P

    Journal: Scientific Reports

    Article Title: Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis

    doi: 10.1038/s41598-018-20316-1

    Figure Lengend Snippet: Effect of clusterin deficiency on TGF-β 1 -induced myofibroblast differentiation and collagen deposition. Lung fibroblasts were transduced with clusterin shRNA (shCLU, open bars) and mock shRNA vectors (grey bars) or remained untransfected (black bars). αSMA mRNA was assessed via qRT-PCR ( A ) and clusterin and αSMA protein levels detected by western blotting ( B,C quantification). 48 h following TGF-β 1 stimulation (40 pM) αSMA mRNA and protein were increased. Basal and increased levels of αSMA mRNA and protein, however, did not vary between clusterin deficient, mock-transduced and control fibroblasts. Collagen mRNA ( D ) and deposition ( E , F quantification) assessed by qRT-PCR and immunofluorescence staining were significantly increased in response to TGF-β 1 . Although, basal and TGF-β 1 induced changes in collagen levels varied between clusterin deficient, mock and control fibroblasts, the overall fold-increase in collagen mRNA and deposition levels did not significantly change between clusterin deficient fibroblasts and control/mock fibroblasts. Data generated in A-F is representative of two individual experiments with fibroblasts derived from 2 donors. Full-length western blots are presented in Supplementary Figure e5 . Scale bar in E represents 10 µm. ** P

    Article Snippet: Lentiviral plasmids with short hairpin sequences targeting clusterin were obtained from GE Healthcare UK.

    Techniques: Transduction, shRNA, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Generated, Derivative Assay

    Localization of clusterin in normal human lung. Clusterin was detected immunohistochemically by staining formalin-fixed, paraffin embedded 3 μm sections of human control lung tissue. Representative images of clusterin (clu, A-C, brown/red, nuclei - blue) and elastic fibers (( D ), grey/black) in tissue obtained from control lung (n = 3). Clusterin localizes to fibroblast-like cells ( A ), to small areas of bronchial epithelial cells ( B ) and to elastic fibers in blood vessels and alveolar walls ( C , D ) serial sections). Clusterin was not detectable in macrophages, alveolar epithelial cells ( A ) or endothelial cells ( C ). Different cell populations/structures are indicated by arrows: f - fibroblast-like cell, m - macrophage, e – alveolar epithelial cell, be - bronchial epithelial cell, en - endothelial cell, smc – smooth muscle cell, ef - elastic fibers. Scale bar represents 25 µm.

    Journal: Scientific Reports

    Article Title: Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis

    doi: 10.1038/s41598-018-20316-1

    Figure Lengend Snippet: Localization of clusterin in normal human lung. Clusterin was detected immunohistochemically by staining formalin-fixed, paraffin embedded 3 μm sections of human control lung tissue. Representative images of clusterin (clu, A-C, brown/red, nuclei - blue) and elastic fibers (( D ), grey/black) in tissue obtained from control lung (n = 3). Clusterin localizes to fibroblast-like cells ( A ), to small areas of bronchial epithelial cells ( B ) and to elastic fibers in blood vessels and alveolar walls ( C , D ) serial sections). Clusterin was not detectable in macrophages, alveolar epithelial cells ( A ) or endothelial cells ( C ). Different cell populations/structures are indicated by arrows: f - fibroblast-like cell, m - macrophage, e – alveolar epithelial cell, be - bronchial epithelial cell, en - endothelial cell, smc – smooth muscle cell, ef - elastic fibers. Scale bar represents 25 µm.

    Article Snippet: Lentiviral plasmids with short hairpin sequences targeting clusterin were obtained from GE Healthcare UK.

    Techniques: Staining, Formalin-fixed Paraffin-Embedded

    Effect of clusterin deficiency on lung fibroblast proliferation.Lung fibroblasts were transduced with clusterin shRNA (shCLU, open bars) and mock shRNA vectors (grey bars) or remained untreated (black bars). Proliferation in shRNA-mediated clusterin deficient fibroblasts ( A ) or fibrotic lung fibroblasts ( B ) compared with controls was assessed in response to the indicated stimuli for 48 h or 72 h for FBS by counting DAPI-positive nuclei in a high-throughput immunofluorescence assay. Cell numbers were normalized to cell counts of controls (0.4% FBS in DMEM) and expressed as percent change in proliferation relative to control (n = 6). Data representatives of two individual experiments with fibroblasts derived from 1 donor per group. Significances compared with controls are marked with (#) symbol and significances between controls vs. shCLU or non-fibrotic vs. fibrotic lung fibroblasts are indicated with (*). */# P

    Journal: Scientific Reports

    Article Title: Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis

    doi: 10.1038/s41598-018-20316-1

    Figure Lengend Snippet: Effect of clusterin deficiency on lung fibroblast proliferation.Lung fibroblasts were transduced with clusterin shRNA (shCLU, open bars) and mock shRNA vectors (grey bars) or remained untreated (black bars). Proliferation in shRNA-mediated clusterin deficient fibroblasts ( A ) or fibrotic lung fibroblasts ( B ) compared with controls was assessed in response to the indicated stimuli for 48 h or 72 h for FBS by counting DAPI-positive nuclei in a high-throughput immunofluorescence assay. Cell numbers were normalized to cell counts of controls (0.4% FBS in DMEM) and expressed as percent change in proliferation relative to control (n = 6). Data representatives of two individual experiments with fibroblasts derived from 1 donor per group. Significances compared with controls are marked with (#) symbol and significances between controls vs. shCLU or non-fibrotic vs. fibrotic lung fibroblasts are indicated with (*). */# P

    Article Snippet: Lentiviral plasmids with short hairpin sequences targeting clusterin were obtained from GE Healthcare UK.

    Techniques: Transduction, shRNA, High Throughput Screening Assay, Immunofluorescence, Derivative Assay

    Characterization of LuloHya. Hyaluronidase activity is female specific in Lu . longipalpis . (A) SGE from 10 male Lu . longipalpis did not show any cleavage of HA in the turbidimetric assay, expressed as the % of remaining HA. On the contrary, SGE from 1 female and 10 nM recombinant LuloHya exhibited remarkable hyaluronidase activity. Multiple comparisons were done by one-way ANOVA (****: p

    Journal: PLoS Pathogens

    Article Title: Immunity to LuloHya and Lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection

    doi: 10.1371/journal.ppat.1007006

    Figure Lengend Snippet: Characterization of LuloHya. Hyaluronidase activity is female specific in Lu . longipalpis . (A) SGE from 10 male Lu . longipalpis did not show any cleavage of HA in the turbidimetric assay, expressed as the % of remaining HA. On the contrary, SGE from 1 female and 10 nM recombinant LuloHya exhibited remarkable hyaluronidase activity. Multiple comparisons were done by one-way ANOVA (****: p

    Article Snippet: LuloHya coding DNA sequence (AF132515) was codon-optimized for mammalian expression and synthesized by BioBasic Inc. VR2001-TOPO vector containing LuloHya sequence (Vical Incorporated) was transformed in One Shot TOP10 Chemically Competent E . coli (Invitrogen).

    Techniques: Activity Assay, Hemagglutination Assay, Turbidimetric Assay, Recombinant

    Validation of gene expression of cytokines and chemokines from HMVEC cells in the presence of LuloHya, Lundep or SGE. RT-PCR for validation of gene expression results obtained with the Human Cytokines Chemokines RT 2 Profiler PCR Array PAHS-150ZD. Specific set of primers were used to amplify (A) CSF2; (B) CSF3; (C) LIF; (D) CXCL1; (E) CXCL2 and (F) CXCL8. Biological triplicates and technical duplicates were analyzed. Negative controls consisted of cDNA isolated from HMVEC cells incubated with incomplete medium. Results are expressed as the fold change of the gene expression normalized against the standard gene HPRT1 (NM_000194). Multiple comparisons were done by one-way ANOVA (****: p

    Journal: PLoS Pathogens

    Article Title: Immunity to LuloHya and Lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection

    doi: 10.1371/journal.ppat.1007006

    Figure Lengend Snippet: Validation of gene expression of cytokines and chemokines from HMVEC cells in the presence of LuloHya, Lundep or SGE. RT-PCR for validation of gene expression results obtained with the Human Cytokines Chemokines RT 2 Profiler PCR Array PAHS-150ZD. Specific set of primers were used to amplify (A) CSF2; (B) CSF3; (C) LIF; (D) CXCL1; (E) CXCL2 and (F) CXCL8. Biological triplicates and technical duplicates were analyzed. Negative controls consisted of cDNA isolated from HMVEC cells incubated with incomplete medium. Results are expressed as the fold change of the gene expression normalized against the standard gene HPRT1 (NM_000194). Multiple comparisons were done by one-way ANOVA (****: p

    Article Snippet: LuloHya coding DNA sequence (AF132515) was codon-optimized for mammalian expression and synthesized by BioBasic Inc. VR2001-TOPO vector containing LuloHya sequence (Vical Incorporated) was transformed in One Shot TOP10 Chemically Competent E . coli (Invitrogen).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Isolation, Incubation

    In vivo recruitment of polymorphonuclear leukocytes by LuloHya, Lundep or SGE in mice. Recruitment of neutrophils to the inoculation site was determined by flow cytometry analysis. ( A ) Gating strategy analysis based on the CD11b expression to identify neutrophils (Ly6G + Ly6C int ) and inflammatory monocytes (Ly6G - Ly6C hi ). Mice (C57BL/6) were intradermally inoculated with LuloHya (10 μg and 1 μg), Lundep (10 μg and 1 μg), Lu . longipalpis SGE (equivalent to 2 SG pairs) and PBS alone as a negative control. Ears injected with 1 μg of non-related salivary protein from Ae . aegypti (ASP) were also included. After 2 h, the ears were collected and processed for flow cytometry analysis. (B) Total count of neutrophils per ear. ( C ) Frequency of neutrophils in mouse ears. Results (of at least 2 independent experiments) are shown as mean +/- SEM. Comparisons with PBS group were done by Mann-Whitney U test (**: p

    Journal: PLoS Pathogens

    Article Title: Immunity to LuloHya and Lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection

    doi: 10.1371/journal.ppat.1007006

    Figure Lengend Snippet: In vivo recruitment of polymorphonuclear leukocytes by LuloHya, Lundep or SGE in mice. Recruitment of neutrophils to the inoculation site was determined by flow cytometry analysis. ( A ) Gating strategy analysis based on the CD11b expression to identify neutrophils (Ly6G + Ly6C int ) and inflammatory monocytes (Ly6G - Ly6C hi ). Mice (C57BL/6) were intradermally inoculated with LuloHya (10 μg and 1 μg), Lundep (10 μg and 1 μg), Lu . longipalpis SGE (equivalent to 2 SG pairs) and PBS alone as a negative control. Ears injected with 1 μg of non-related salivary protein from Ae . aegypti (ASP) were also included. After 2 h, the ears were collected and processed for flow cytometry analysis. (B) Total count of neutrophils per ear. ( C ) Frequency of neutrophils in mouse ears. Results (of at least 2 independent experiments) are shown as mean +/- SEM. Comparisons with PBS group were done by Mann-Whitney U test (**: p

    Article Snippet: LuloHya coding DNA sequence (AF132515) was codon-optimized for mammalian expression and synthesized by BioBasic Inc. VR2001-TOPO vector containing LuloHya sequence (Vical Incorporated) was transformed in One Shot TOP10 Chemically Competent E . coli (Invitrogen).

    Techniques: In Vivo, Mouse Assay, Flow Cytometry, Cytometry, Expressing, Negative Control, Injection, MANN-WHITNEY

    Three-dimensional modeling of LuloHya. Predicted 3D model of LuloHya was generated automatically by I-TASSER software. LuloHya coordinates used to generate the model were based on the ligand-free honeybee venom hyaluronidase (1FCQ) and in complex (1FCV) with hyaluronic acid tetramer. (A) Cartoon generated with PyMol shows LuloHya as a typical globular protein with a catalytic site topology of the cleft/groove class. Conserved amino acid residues aspartic acid (D100) and glutamic acid (E102) are highlighted in red. (B) Electrostatic potential of LuloHya generated by the APBS tools shows charged amino acids evenly distributed on the surface of the enzyme as determined by the Adaptive Poisson-Bolzmann Solver software with blue being positive and red being negative. Predicted substrate (HA tetramer) bound to LuloHya catalytic cleft if shown.

    Journal: PLoS Pathogens

    Article Title: Immunity to LuloHya and Lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection

    doi: 10.1371/journal.ppat.1007006

    Figure Lengend Snippet: Three-dimensional modeling of LuloHya. Predicted 3D model of LuloHya was generated automatically by I-TASSER software. LuloHya coordinates used to generate the model were based on the ligand-free honeybee venom hyaluronidase (1FCQ) and in complex (1FCV) with hyaluronic acid tetramer. (A) Cartoon generated with PyMol shows LuloHya as a typical globular protein with a catalytic site topology of the cleft/groove class. Conserved amino acid residues aspartic acid (D100) and glutamic acid (E102) are highlighted in red. (B) Electrostatic potential of LuloHya generated by the APBS tools shows charged amino acids evenly distributed on the surface of the enzyme as determined by the Adaptive Poisson-Bolzmann Solver software with blue being positive and red being negative. Predicted substrate (HA tetramer) bound to LuloHya catalytic cleft if shown.

    Article Snippet: LuloHya coding DNA sequence (AF132515) was codon-optimized for mammalian expression and synthesized by BioBasic Inc. VR2001-TOPO vector containing LuloHya sequence (Vical Incorporated) was transformed in One Shot TOP10 Chemically Competent E . coli (Invitrogen).

    Techniques: Generated, Software, Hemagglutination Assay

    Anti-LuloHya antibodies block the hyaluronidase activity of SGE and LuloHya. Hyaluronidase activity of Lu . longipalpis SGE and 10 nM LuloHya in the presence of 5 μg of rabbit IgG-control (pre-immune IgG) or 5 μg of rabbit anti-LuloHya IgG. As negative controls, TBS was added instead of SGE or recombinant protein. Biological triplicates were tested. Bars indicate SEM.

    Journal: PLoS Pathogens

    Article Title: Immunity to LuloHya and Lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection

    doi: 10.1371/journal.ppat.1007006

    Figure Lengend Snippet: Anti-LuloHya antibodies block the hyaluronidase activity of SGE and LuloHya. Hyaluronidase activity of Lu . longipalpis SGE and 10 nM LuloHya in the presence of 5 μg of rabbit IgG-control (pre-immune IgG) or 5 μg of rabbit anti-LuloHya IgG. As negative controls, TBS was added instead of SGE or recombinant protein. Biological triplicates were tested. Bars indicate SEM.

    Article Snippet: LuloHya coding DNA sequence (AF132515) was codon-optimized for mammalian expression and synthesized by BioBasic Inc. VR2001-TOPO vector containing LuloHya sequence (Vical Incorporated) was transformed in One Shot TOP10 Chemically Competent E . coli (Invitrogen).

    Techniques: Blocking Assay, Activity Assay, Recombinant

    LuloHya and Lundep enhance dermonecrotic lesions caused by HF injection. (A) Ears of animals injected intradermally with 3 μg of HF along with SGE (equivalent to 2 SG), 10 μg of LuloHya, 10 μg of Lundep or a combination of 5 μg of LuloHya and Lundep (HF column) showed larger lesions than ears injected with HF and PBS. Left column pictures (PBS) show mice ears injected with SGE, LuloHya, Lundep and the combination of both proteins in the absence of HF. Two hours after injection, ears were excised and measurements of the hemorrhage area were taken. (B) D * d * π 4 where D is the longest and d the smallest lesion diameter. Multiple comparisons were done by one-way ANOVA (****: p

    Journal: PLoS Pathogens

    Article Title: Immunity to LuloHya and Lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection

    doi: 10.1371/journal.ppat.1007006

    Figure Lengend Snippet: LuloHya and Lundep enhance dermonecrotic lesions caused by HF injection. (A) Ears of animals injected intradermally with 3 μg of HF along with SGE (equivalent to 2 SG), 10 μg of LuloHya, 10 μg of Lundep or a combination of 5 μg of LuloHya and Lundep (HF column) showed larger lesions than ears injected with HF and PBS. Left column pictures (PBS) show mice ears injected with SGE, LuloHya, Lundep and the combination of both proteins in the absence of HF. Two hours after injection, ears were excised and measurements of the hemorrhage area were taken. (B) D * d * π 4 where D is the longest and d the smallest lesion diameter. Multiple comparisons were done by one-way ANOVA (****: p

    Article Snippet: LuloHya coding DNA sequence (AF132515) was codon-optimized for mammalian expression and synthesized by BioBasic Inc. VR2001-TOPO vector containing LuloHya sequence (Vical Incorporated) was transformed in One Shot TOP10 Chemically Competent E . coli (Invitrogen).

    Techniques: Injection, Mouse Assay

    Vaccination studies with LuloHya and Lundep against L . major infection. (A) Lesion size of C57BL/6 mice due to L . major infection steadily increased from the second week of the follow-up period until it stabilized or started to decrease after week 7 in all animals. For control animals (only immunized with Magic Mouse Adjuvant) lesion size was higher than vaccinated groups from week 4 onwards. The symbols represent the lesion size mean of 10 ears ± SEM analyzed by analysis of variance. (B) Lesion size values of C57BL/6 mice were converted to the area under the curve (AUC) showing that mice immunized against either LuloHya and Lundep presented significantly reduced lesions. (C) Parasite load of ears from C57BL/6 mice vaccinated with LuloHya or Lundep was significantly lower than control group (P

    Journal: PLoS Pathogens

    Article Title: Immunity to LuloHya and Lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection

    doi: 10.1371/journal.ppat.1007006

    Figure Lengend Snippet: Vaccination studies with LuloHya and Lundep against L . major infection. (A) Lesion size of C57BL/6 mice due to L . major infection steadily increased from the second week of the follow-up period until it stabilized or started to decrease after week 7 in all animals. For control animals (only immunized with Magic Mouse Adjuvant) lesion size was higher than vaccinated groups from week 4 onwards. The symbols represent the lesion size mean of 10 ears ± SEM analyzed by analysis of variance. (B) Lesion size values of C57BL/6 mice were converted to the area under the curve (AUC) showing that mice immunized against either LuloHya and Lundep presented significantly reduced lesions. (C) Parasite load of ears from C57BL/6 mice vaccinated with LuloHya or Lundep was significantly lower than control group (P

    Article Snippet: LuloHya coding DNA sequence (AF132515) was codon-optimized for mammalian expression and synthesized by BioBasic Inc. VR2001-TOPO vector containing LuloHya sequence (Vical Incorporated) was transformed in One Shot TOP10 Chemically Competent E . coli (Invitrogen).

    Techniques: Infection, Mouse Assay

    Specificity of hyaluronidase activity of LuloHya. (A) Five micrograms of high molecular weight HA (H), chondroitin sulfate B (CS), dextran sulfate (DS) and heparin (Hep) were fractionated on a 1.2% agarose gel alone or after incubation with LuloHya or Lu . longipalpis SGE. As molecular weight markers, Select-HA HiLadder, Select-HA 250k (Hyalose) and GeneRuler 1kb DNA ladder were used (Thermo Scientific). (B) Five micrograms of high, medium and low molecular weight HA (H, M and L, respectively) were separated on a 1.2% agarose gel alone of after incubation with LuloHya, bovine hyaluronidase (BovHya), Streptomyces hyalurolyticus hyaluronidase (BactHya) and Lu . longipalpis SGE.

    Journal: PLoS Pathogens

    Article Title: Immunity to LuloHya and Lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection

    doi: 10.1371/journal.ppat.1007006

    Figure Lengend Snippet: Specificity of hyaluronidase activity of LuloHya. (A) Five micrograms of high molecular weight HA (H), chondroitin sulfate B (CS), dextran sulfate (DS) and heparin (Hep) were fractionated on a 1.2% agarose gel alone or after incubation with LuloHya or Lu . longipalpis SGE. As molecular weight markers, Select-HA HiLadder, Select-HA 250k (Hyalose) and GeneRuler 1kb DNA ladder were used (Thermo Scientific). (B) Five micrograms of high, medium and low molecular weight HA (H, M and L, respectively) were separated on a 1.2% agarose gel alone of after incubation with LuloHya, bovine hyaluronidase (BovHya), Streptomyces hyalurolyticus hyaluronidase (BactHya) and Lu . longipalpis SGE.

    Article Snippet: LuloHya coding DNA sequence (AF132515) was codon-optimized for mammalian expression and synthesized by BioBasic Inc. VR2001-TOPO vector containing LuloHya sequence (Vical Incorporated) was transformed in One Shot TOP10 Chemically Competent E . coli (Invitrogen).

    Techniques: Activity Assay, Molecular Weight, Hemagglutination Assay, Agarose Gel Electrophoresis, Incubation

    Biochemical characterization of the recombinant protein LuloHya. (A) Purification of LuloHya by size exclusion chromatography using Superdex 200 Increase 10/300 GL column. (B) Coomassie-stained gel electrophoresis of LuloHya (1 μg). Mouse anti-LuloHya antibodies (1:5,000) recognized LuloHya (100 ng) and a single band in the SGE (5 pairs of SG) by Western blot. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (C) NuPAGE Novex 4–12% Bis-Tris protein gel shows differences in electrophoretic pattern of LuloHya (1 μg) and its deglycosylated form (deLuloHya: 1 μg) which runs at the expected molecular weight (42.3 kDa). M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (D) Hyaluronidase activity of 10 nM LuloHya and its deglycosylated form (deLuloHya). As negative controls, 4 micrograms of HA were incubated with TBS instead of recombinant protein or the deglycosylation enzyme mix (DeglycoMx Kit, QABio) without protein. (E) Turbidimetric assay showed a clear pH dependence of hyaluronidase activity of LuloHya. Reaction mixtures were prepared with solutions containing 25 mM buffer (described in Methods), 100 mM NaCl, 0.1% BSA, and different pH values (4–12.5; adjusted with a pHmeter 430, Corning). (F) Ionic strength dependency was analyzed in reaction mixtures of 25 mM HEPES, 0.1% BSA, pH 7.3 with variable NaCl concentration (25–1000 mM). Hyaluronidase activity is inversely expressed as the remaining HA (%) after treatment of 4 μg of HA with 10 nM enzyme during 1 h at 37°C. Biological triplicates and technical duplicates were assessed. Multiple comparisons were done by one-way ANOVA (****: p

    Journal: PLoS Pathogens

    Article Title: Immunity to LuloHya and Lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection

    doi: 10.1371/journal.ppat.1007006

    Figure Lengend Snippet: Biochemical characterization of the recombinant protein LuloHya. (A) Purification of LuloHya by size exclusion chromatography using Superdex 200 Increase 10/300 GL column. (B) Coomassie-stained gel electrophoresis of LuloHya (1 μg). Mouse anti-LuloHya antibodies (1:5,000) recognized LuloHya (100 ng) and a single band in the SGE (5 pairs of SG) by Western blot. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (C) NuPAGE Novex 4–12% Bis-Tris protein gel shows differences in electrophoretic pattern of LuloHya (1 μg) and its deglycosylated form (deLuloHya: 1 μg) which runs at the expected molecular weight (42.3 kDa). M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (D) Hyaluronidase activity of 10 nM LuloHya and its deglycosylated form (deLuloHya). As negative controls, 4 micrograms of HA were incubated with TBS instead of recombinant protein or the deglycosylation enzyme mix (DeglycoMx Kit, QABio) without protein. (E) Turbidimetric assay showed a clear pH dependence of hyaluronidase activity of LuloHya. Reaction mixtures were prepared with solutions containing 25 mM buffer (described in Methods), 100 mM NaCl, 0.1% BSA, and different pH values (4–12.5; adjusted with a pHmeter 430, Corning). (F) Ionic strength dependency was analyzed in reaction mixtures of 25 mM HEPES, 0.1% BSA, pH 7.3 with variable NaCl concentration (25–1000 mM). Hyaluronidase activity is inversely expressed as the remaining HA (%) after treatment of 4 μg of HA with 10 nM enzyme during 1 h at 37°C. Biological triplicates and technical duplicates were assessed. Multiple comparisons were done by one-way ANOVA (****: p

    Article Snippet: LuloHya coding DNA sequence (AF132515) was codon-optimized for mammalian expression and synthesized by BioBasic Inc. VR2001-TOPO vector containing LuloHya sequence (Vical Incorporated) was transformed in One Shot TOP10 Chemically Competent E . coli (Invitrogen).

    Techniques: Recombinant, Purification, Size-exclusion Chromatography, Staining, Nucleic Acid Electrophoresis, Western Blot, Molecular Weight, Activity Assay, Hemagglutination Assay, Incubation, Turbidimetric Assay, Concentration Assay

    MT1-MMP overexpression inhibited the protrusive morphology of MDA-MB 231 breast cancer cells in 3D culture. a ( top ) MDA-MB 231 MT1-MMP cells were embedded in Matrigel and imaged every day for 5 days at 10× magnification. Shown are representative fields of view of each cell line at day 5 and a respective inset. Red arrow shows a portion of the protrusive network MDA-MB 231 cells form in 3D culture. White arrows show MDA-MB 231 MT1-MMP cell colonies that have retained circularity after 5 days in 3D culture. Scale bars = 100 μm. ( bottom ) Five z-stacks per cell line were acquired every day for 5 days and disseminations and protrusions were quantified per colony for each cell line. b Representative 3D volume views of immunofluorescence analysis after MDA-MB 231 MT1-MMP cells were embedded in Matrigel for 5 days. Samples were imaged using confocal microscopy at 60× magnification and are displayed as overlays showing MT1-MMP signal ( green ), DAPI ( blue ) and Alexa633 phalloidin ( red ) channels. Scale bars = 100 μm. Red arrow shows protrusive MDA-MB 231 cells, whereas green arrows show circular colonies in MDA-MB 231 MT1-MMP cell lines that are positive for MT1-MMP protein signal. Scale bars = 100 μm

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MT1-MMP overexpression inhibited the protrusive morphology of MDA-MB 231 breast cancer cells in 3D culture. a ( top ) MDA-MB 231 MT1-MMP cells were embedded in Matrigel and imaged every day for 5 days at 10× magnification. Shown are representative fields of view of each cell line at day 5 and a respective inset. Red arrow shows a portion of the protrusive network MDA-MB 231 cells form in 3D culture. White arrows show MDA-MB 231 MT1-MMP cell colonies that have retained circularity after 5 days in 3D culture. Scale bars = 100 μm. ( bottom ) Five z-stacks per cell line were acquired every day for 5 days and disseminations and protrusions were quantified per colony for each cell line. b Representative 3D volume views of immunofluorescence analysis after MDA-MB 231 MT1-MMP cells were embedded in Matrigel for 5 days. Samples were imaged using confocal microscopy at 60× magnification and are displayed as overlays showing MT1-MMP signal ( green ), DAPI ( blue ) and Alexa633 phalloidin ( red ) channels. Scale bars = 100 μm. Red arrow shows protrusive MDA-MB 231 cells, whereas green arrows show circular colonies in MDA-MB 231 MT1-MMP cell lines that are positive for MT1-MMP protein signal. Scale bars = 100 μm

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Over Expression, Multiple Displacement Amplification, Immunofluorescence, Confocal Microscopy

    MT1-MMP overexpression in MCF-7 cells induced loss of colony organization and was inversely correlated with a protrusive morphology in 3D culture. a ( top ) MCF-7 MT1-MMP cells were embedded in Matrigel and imaged every day for 5 days at 10× magnification. Shown is a representative field of view of each cell line at day 5, and indicated inset images which show the cell features quantified: Circular colonies ( white arrow ), disseminations around colonies ( green arrow ), or protrusions emanating from colonies ( red arrows ). Scale bars = 100 μm. ( bottom ) Five z-stacks per cell line were acquired every day for 5 days and disseminations and protrusions were quantified per colony for each cell line. b Representative 3D volume views of immunofluorescence analysis after MCF-7 MT1-MMP cells were embedded in Matrigel for 5 days. Samples were imaged using confocal microscopy at 60× and are displayed as overlays showing MT1-MMP signal ( green ), DAPI ( blue ) and Alexa633 phalloidin ( red ) channels. Scale bars = 100 μm. White arrow show circular colonies. Green arrows displays single cells that disseminated from the nearby colonies and show MT1-MMP protein. Red arrow shows an F-actin protrusion emanating from a circular colony ( c ) Single cells, F-actin disseminations, F-actin protrusions, and zsGreen protrusions were quantified from 20× magnification 3D volumes acquired after MCF-7 MT1-MMP cell lines stably expressing zsGreen were embedded in Matrigel for 5 days. The 60× magnification 3D volume of MT1-MMP C3 cells shows both F-actin ( red arrow ) and zsGreen ( blue arrow ) protrusions emerging from a colony. Scale bars = 100 μm

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MT1-MMP overexpression in MCF-7 cells induced loss of colony organization and was inversely correlated with a protrusive morphology in 3D culture. a ( top ) MCF-7 MT1-MMP cells were embedded in Matrigel and imaged every day for 5 days at 10× magnification. Shown is a representative field of view of each cell line at day 5, and indicated inset images which show the cell features quantified: Circular colonies ( white arrow ), disseminations around colonies ( green arrow ), or protrusions emanating from colonies ( red arrows ). Scale bars = 100 μm. ( bottom ) Five z-stacks per cell line were acquired every day for 5 days and disseminations and protrusions were quantified per colony for each cell line. b Representative 3D volume views of immunofluorescence analysis after MCF-7 MT1-MMP cells were embedded in Matrigel for 5 days. Samples were imaged using confocal microscopy at 60× and are displayed as overlays showing MT1-MMP signal ( green ), DAPI ( blue ) and Alexa633 phalloidin ( red ) channels. Scale bars = 100 μm. White arrow show circular colonies. Green arrows displays single cells that disseminated from the nearby colonies and show MT1-MMP protein. Red arrow shows an F-actin protrusion emanating from a circular colony ( c ) Single cells, F-actin disseminations, F-actin protrusions, and zsGreen protrusions were quantified from 20× magnification 3D volumes acquired after MCF-7 MT1-MMP cell lines stably expressing zsGreen were embedded in Matrigel for 5 days. The 60× magnification 3D volume of MT1-MMP C3 cells shows both F-actin ( red arrow ) and zsGreen ( blue arrow ) protrusions emerging from a colony. Scale bars = 100 μm

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Over Expression, Immunofluorescence, Confocal Microscopy, Stable Transfection, Expressing

    Low levels of MT1-MMP expression increased survivability of MCF-7 breast cancer cells to serum-free stress. a Viability of MCF-7 MT1-MMP cell lines during incubation in media containing 10 % FBS ( top ) or serum free media ( bottom ) measured daily for 7 days using Celltiter96®. b Viability of MCF-7 MT1-MMP cell lines and C3 cell line variants during incubation in SF media measured every 3 days for 9 days. Immunoblot analysis ( bottom ) shows PH3 protein levels collected from MCF-7 MT1-MMP cell lines and C3 cell line variants incubated in SF media for 6 days. β-actin was used as a loading control. Bar graph ( right ) shows densitometry analysis of PH3 levels. c Viability of MCF-7 MT1-MMP cell lines was measured after incubation for 6 days in SF media containing increasing concentrations of U0126, BB94, a furin inhibitor or an AKT inhibitor. Black asterisks show statistically significant differences between the initial and day 6 viability within cell lines, and also differences between the day 6 viability of MCF-7 cells compared to MT1-MMP expressing cell lines. Red asterisks indicate significant differences ( p ≤ 0.05) within cell lines between the day 6 viability in SF media compared to the viability after 6 days of incubation with the different concentrations of the inhibitors

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: Low levels of MT1-MMP expression increased survivability of MCF-7 breast cancer cells to serum-free stress. a Viability of MCF-7 MT1-MMP cell lines during incubation in media containing 10 % FBS ( top ) or serum free media ( bottom ) measured daily for 7 days using Celltiter96®. b Viability of MCF-7 MT1-MMP cell lines and C3 cell line variants during incubation in SF media measured every 3 days for 9 days. Immunoblot analysis ( bottom ) shows PH3 protein levels collected from MCF-7 MT1-MMP cell lines and C3 cell line variants incubated in SF media for 6 days. β-actin was used as a loading control. Bar graph ( right ) shows densitometry analysis of PH3 levels. c Viability of MCF-7 MT1-MMP cell lines was measured after incubation for 6 days in SF media containing increasing concentrations of U0126, BB94, a furin inhibitor or an AKT inhibitor. Black asterisks show statistically significant differences between the initial and day 6 viability within cell lines, and also differences between the day 6 viability of MCF-7 cells compared to MT1-MMP expressing cell lines. Red asterisks indicate significant differences ( p ≤ 0.05) within cell lines between the day 6 viability in SF media compared to the viability after 6 days of incubation with the different concentrations of the inhibitors

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Expressing, Incubation

    Overexpression of MT1-MMP in MDA-MB 231 cells negatively affected migration and viability ( a ) Immunoblot analysis (AB6004) showing pro-, active, and degradation forms of MT1-MMP in MDA-MB 231 breast cancer cells and three MDA-MB 231 cell lines expressing different levels of MT1-MMP . β-actin was used as a loading control. b Transwell migration assay of MDA-MB 231 MT1-MMP cells incubated in SF media for 12 h. c Viability of MDA-MB 231 MT1-MMP cell lines during incubation in media containing 10 % FBS ( top ) or serum free media ( bottom ) measured daily for 7 days using Celltiter96®

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: Overexpression of MT1-MMP in MDA-MB 231 cells negatively affected migration and viability ( a ) Immunoblot analysis (AB6004) showing pro-, active, and degradation forms of MT1-MMP in MDA-MB 231 breast cancer cells and three MDA-MB 231 cell lines expressing different levels of MT1-MMP . β-actin was used as a loading control. b Transwell migration assay of MDA-MB 231 MT1-MMP cells incubated in SF media for 12 h. c Viability of MDA-MB 231 MT1-MMP cell lines during incubation in media containing 10 % FBS ( top ) or serum free media ( bottom ) measured daily for 7 days using Celltiter96®

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Over Expression, Multiple Displacement Amplification, Migration, Expressing, Transwell Migration Assay, Incubation

    Schematic overview of MT1-MMP expression levels and associated changes in substrate degradation and cell migration in 2D culture, phenotypes in 3D culture, and tumourigenesis in vivo. Schematic representation of the findings of this study showing cell phenotypes across 2D and 3D culture platforms and in vivo. Legend describing molecular components in diagrams is shown at the top, and fold change relative to MCF-7 parental cells is in the brackets to the right of the bolded titles. MT1-MMP deficient breast cancer cells, such as MCF-7 cells, are incapable of proMMP-2 activation or ECM degradation, and show low migration and viability during serum-free incubation. These cells retain a circular morphology in 3D culture, and do not form vascularized tumours nor display high extravasation efficiency in vivo. Cells expressing high levels of MT1-MMP are capable of proMMP-2 activation and widespread ECM degradation, have increased survivability to serum-free stress, but do not demonstrate increased migration in 2D experiments. In 3D culture, these cells demonstrate a dissemination morphology and cell fragment release mediated by MT1-MMP. Despite MT1-MMP protein production and associated substrate degradation, these cells are unable to form vascularized tumours or increase their extravasation efficiency in vivo. Cells expressing low levels of MT1-MMP do not demonstrate proMMP-2 activation or widespread ECM degradation, but do show increased migratory potential, and high viability during serum-free incubation. These cells demonstrate a protrusive morphology in 3D culture, form vascularized tumours in vivo, and have significantly increased extravasation efficiency. Data figures within this study that correspond to the diagrams within this model are in red text

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: Schematic overview of MT1-MMP expression levels and associated changes in substrate degradation and cell migration in 2D culture, phenotypes in 3D culture, and tumourigenesis in vivo. Schematic representation of the findings of this study showing cell phenotypes across 2D and 3D culture platforms and in vivo. Legend describing molecular components in diagrams is shown at the top, and fold change relative to MCF-7 parental cells is in the brackets to the right of the bolded titles. MT1-MMP deficient breast cancer cells, such as MCF-7 cells, are incapable of proMMP-2 activation or ECM degradation, and show low migration and viability during serum-free incubation. These cells retain a circular morphology in 3D culture, and do not form vascularized tumours nor display high extravasation efficiency in vivo. Cells expressing high levels of MT1-MMP are capable of proMMP-2 activation and widespread ECM degradation, have increased survivability to serum-free stress, but do not demonstrate increased migration in 2D experiments. In 3D culture, these cells demonstrate a dissemination morphology and cell fragment release mediated by MT1-MMP. Despite MT1-MMP protein production and associated substrate degradation, these cells are unable to form vascularized tumours or increase their extravasation efficiency in vivo. Cells expressing low levels of MT1-MMP do not demonstrate proMMP-2 activation or widespread ECM degradation, but do show increased migratory potential, and high viability during serum-free incubation. These cells demonstrate a protrusive morphology in 3D culture, form vascularized tumours in vivo, and have significantly increased extravasation efficiency. Data figures within this study that correspond to the diagrams within this model are in red text

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Expressing, Migration, In Vivo, Activation Assay, Incubation

    MT1-MMP activity is inversely correlated to the migratory potential of MCF-7 breast cancer cells ( a ) MCF-7 MT1-MMP cell lines stably expressing zsGreen were seeded on Alexa594-gelatin coated coverlips in media containing 0.1 % DMSO (control) or 10 μm BB94 and incubated in a live imaging chamber. Each sample was imaged at the same five stage positions every 10 mins for 20 h to visualize zsGreen cell movement and associated ECM degradation (Additional files 3, 4, 5 and 6). Shown are stills of the Alexa594 gelatin channel and an overlay including the zsGreen cells at time 0 and 20 h post-seeding of the control sample (BB94 not shown). Scale bars = 100 μm. b Time-lapse videos from ( a ) were analyzed using the ADAPT plugin for ImageJ and all individual cells tracked from each cell line were examined and grouped according to their migration distance from initial point of tracking. c Percentage of cells per field of view from each cell line that degraded the underlying AlexaFluour594-gelatin at five different time points

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MT1-MMP activity is inversely correlated to the migratory potential of MCF-7 breast cancer cells ( a ) MCF-7 MT1-MMP cell lines stably expressing zsGreen were seeded on Alexa594-gelatin coated coverlips in media containing 0.1 % DMSO (control) or 10 μm BB94 and incubated in a live imaging chamber. Each sample was imaged at the same five stage positions every 10 mins for 20 h to visualize zsGreen cell movement and associated ECM degradation (Additional files 3, 4, 5 and 6). Shown are stills of the Alexa594 gelatin channel and an overlay including the zsGreen cells at time 0 and 20 h post-seeding of the control sample (BB94 not shown). Scale bars = 100 μm. b Time-lapse videos from ( a ) were analyzed using the ADAPT plugin for ImageJ and all individual cells tracked from each cell line were examined and grouped according to their migration distance from initial point of tracking. c Percentage of cells per field of view from each cell line that degraded the underlying AlexaFluour594-gelatin at five different time points

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Activity Assay, Stable Transfection, Expressing, Incubation, Imaging, Migration

    Metastatic human 21 T breast cancer cells showed undetectable levels of MT1-MMP protein similar to MCF-7 C3 cells. Protein lysate from human 21 T breast cancer cell lines, which represent a progression series from atypical ductal hyperplasia (21PT-ADH), to ductal carcinoma in situ (21NT – DCIS), to invasive mammary carcinoma (21MT-1- IMC), were analyzed via immunoblot for MT1-MMP protein levels along with the MCF-7 MT1-MMP cell lines. The blots were probed with either AB6004 ( top ) or AB51074 ( bottom ) and shown as the normal exposure and as transformed versions to clearly show banding pattern. Asterisks indicate MT1-MMP isoforms (green – pro form, red- active form, orange – degradation forms). β-actin was used as a loading control

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: Metastatic human 21 T breast cancer cells showed undetectable levels of MT1-MMP protein similar to MCF-7 C3 cells. Protein lysate from human 21 T breast cancer cell lines, which represent a progression series from atypical ductal hyperplasia (21PT-ADH), to ductal carcinoma in situ (21NT – DCIS), to invasive mammary carcinoma (21MT-1- IMC), were analyzed via immunoblot for MT1-MMP protein levels along with the MCF-7 MT1-MMP cell lines. The blots were probed with either AB6004 ( top ) or AB51074 ( bottom ) and shown as the normal exposure and as transformed versions to clearly show banding pattern. Asterisks indicate MT1-MMP isoforms (green – pro form, red- active form, orange – degradation forms). β-actin was used as a loading control

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: In Situ, Transformation Assay

    MCF-7 MT1-MMP C3 cells displayed high tumorigenic potential when implanted onto the avian embryo CAM. MCF-7 MT1-MMP cell lines and MDA-MB 231 cells stably expressing zsGreen were implanted into the CAM of day 9 ex ovo chicken embryos and visualized 8 days post –implantation using a fluorescence stereoscope to analyze tumor vascularization. Displayed are representative bright field images showing the area of implantation on the embryo, and respective fluorescent images showing the zsGreen channel. The white boxes outline the insets showing vascularization of the MT1-MMP C3 and MDA-MB 231 tumours. Bar graph shows percentage of tumours that were vascularized ( N ≥ 11). Scale bars = 2 mm

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MCF-7 MT1-MMP C3 cells displayed high tumorigenic potential when implanted onto the avian embryo CAM. MCF-7 MT1-MMP cell lines and MDA-MB 231 cells stably expressing zsGreen were implanted into the CAM of day 9 ex ovo chicken embryos and visualized 8 days post –implantation using a fluorescence stereoscope to analyze tumor vascularization. Displayed are representative bright field images showing the area of implantation on the embryo, and respective fluorescent images showing the zsGreen channel. The white boxes outline the insets showing vascularization of the MT1-MMP C3 and MDA-MB 231 tumours. Bar graph shows percentage of tumours that were vascularized ( N ≥ 11). Scale bars = 2 mm

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Chick Chorioallantoic Membrane Assay, Multiple Displacement Amplification, Stable Transfection, Expressing, Fluorescence

    MT1-MMP expression does not correlate with increased migratory potential of breast cancer cells. a qPCR analysis of MT1-MMP mRNA levels from MCF-7, MDA-MB 231, and HS578t breast cancer cells. b Immunoblot (AB51074) and reverse zymography analysis comparing MT1-MMP, phospho-ERK and TIMP-2 protein levels between MCF-7, MDA-MB 231, and HS578t breast cancer cells. β-actin and total ERK1/2 were used as loading controls. c Gelatin zymography analysis of MCF-7, MDA-MB 231, and HS578t breast cancer cells incubated in SF media for 12 h. Lane 1 shows proMMP-2 CM activated by MCF-7 C2 cells treated with TIMP-2 CM diluted 1:100 to show proMMP-2 activation as a result of TIMP-2/MT1-MMP. d Transwell migration assay of MCF-7, MDA-MB 231, and HS578t breast cancer cells incubated in SF media for 24 h

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MT1-MMP expression does not correlate with increased migratory potential of breast cancer cells. a qPCR analysis of MT1-MMP mRNA levels from MCF-7, MDA-MB 231, and HS578t breast cancer cells. b Immunoblot (AB51074) and reverse zymography analysis comparing MT1-MMP, phospho-ERK and TIMP-2 protein levels between MCF-7, MDA-MB 231, and HS578t breast cancer cells. β-actin and total ERK1/2 were used as loading controls. c Gelatin zymography analysis of MCF-7, MDA-MB 231, and HS578t breast cancer cells incubated in SF media for 12 h. Lane 1 shows proMMP-2 CM activated by MCF-7 C2 cells treated with TIMP-2 CM diluted 1:100 to show proMMP-2 activation as a result of TIMP-2/MT1-MMP. d Transwell migration assay of MCF-7, MDA-MB 231, and HS578t breast cancer cells incubated in SF media for 24 h

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Zymography, Incubation, Activation Assay, Transwell Migration Assay

    Transient overexpression of MT1-MMP in MCF-7 cells did not result in increased migration and invasion ( a ) qPCR, immunoblot, and gelatin zymography analysis of MT1-MMP mRNA, protein levels, and proMMP-2 activation ability, respectively, of MCF-7 breast cancer cells transiently transfected with MT1-MMP compared to mock transfected cells (control). Immunoblot analysis (AB6004) showed pro-, active, and degradation forms of MT1-MMP protein in MT1-MMP transfected MCF-7 cells. β-actin was used as a loading control. Gelatin zymography analysis showed that MCF-7 cells transiently transfected with MT1-MMP were capable of activating proMMP-2 after 24 h of incubation as shown by intermediate and active forms of MMP-2. b Gelatin zymography analysis of MCF-7 cells transiently transfected with MT1-MMP and incubated for 12 h with serum-free media (SF, top gel) or MMP-2 conditioned media (CM, bottom gel). Lanes 1 and 2: Controls showing proMMP-2 CM chemically activated by APMA. Lanes 4 and 6: Recombinant TIMP-2 (rTIMP-2) was added at 100 ng/ml to enhance MT1-MMP-mediated proMMP-2 activation. c Immunoblot analysis of MT1-MMP transfected cells showing phospho-ERK1/2 levels. Total ERK1/2 was used as a loading control. d Transwell migration and invasion assays of MCF-7 cells transiently transfected with MT1-MMP. Number of migrated/invaded cells were normalized to control MCF-7 cells and expressed as a mean percentage ± SEM. (ns, p > 0.05 by student’s t -test)

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: Transient overexpression of MT1-MMP in MCF-7 cells did not result in increased migration and invasion ( a ) qPCR, immunoblot, and gelatin zymography analysis of MT1-MMP mRNA, protein levels, and proMMP-2 activation ability, respectively, of MCF-7 breast cancer cells transiently transfected with MT1-MMP compared to mock transfected cells (control). Immunoblot analysis (AB6004) showed pro-, active, and degradation forms of MT1-MMP protein in MT1-MMP transfected MCF-7 cells. β-actin was used as a loading control. Gelatin zymography analysis showed that MCF-7 cells transiently transfected with MT1-MMP were capable of activating proMMP-2 after 24 h of incubation as shown by intermediate and active forms of MMP-2. b Gelatin zymography analysis of MCF-7 cells transiently transfected with MT1-MMP and incubated for 12 h with serum-free media (SF, top gel) or MMP-2 conditioned media (CM, bottom gel). Lanes 1 and 2: Controls showing proMMP-2 CM chemically activated by APMA. Lanes 4 and 6: Recombinant TIMP-2 (rTIMP-2) was added at 100 ng/ml to enhance MT1-MMP-mediated proMMP-2 activation. c Immunoblot analysis of MT1-MMP transfected cells showing phospho-ERK1/2 levels. Total ERK1/2 was used as a loading control. d Transwell migration and invasion assays of MCF-7 cells transiently transfected with MT1-MMP. Number of migrated/invaded cells were normalized to control MCF-7 cells and expressed as a mean percentage ± SEM. (ns, p > 0.05 by student’s t -test)

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Over Expression, Migration, Real-time Polymerase Chain Reaction, Zymography, Activation Assay, Transfection, Incubation, Recombinant

    MCF-7 cell lines producing high levels of MT1-MMP protein demonstrated TIMP-2-mediated proMMP-2 activation ( a ) qPCR analysis of MT1-MMP mRNA from MCF-7 MT1-MMP cells lines that stably express different levels of MT1-MMP. Different letters indicate significant differences at p ≤ 0.05 by one-way ANOVA, Tukey’s post-hoc test. b Immunoblot analysis (AB51074) showing pro-, active, and degradation forms of MT1-MMP protein in MCF-7 MT1-MMP cell lines. β-actin was used as a loading control. c Gelatin zymography analysis of MCF-7 MT1-MMP cell lines incubated for 6 or 12 h with either MMP-2 CM alone, or in combination with rTIMP-2 at 100 ng/ml, or rTIMP-2 and BB94 (10 μm). Cells were also incubated for 12 h in MMP-2 CM supplemented with TIMP-2 CM diluted 1:100 in SF media ( bottom ). Bar graph shows densitometry quantification of MMP-2 isoforms from representative zymography of MCF-7 MT1-MMP cell lines incubated with MMP-2 CM and TIMP-2 CM diluted 1:100

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MCF-7 cell lines producing high levels of MT1-MMP protein demonstrated TIMP-2-mediated proMMP-2 activation ( a ) qPCR analysis of MT1-MMP mRNA from MCF-7 MT1-MMP cells lines that stably express different levels of MT1-MMP. Different letters indicate significant differences at p ≤ 0.05 by one-way ANOVA, Tukey’s post-hoc test. b Immunoblot analysis (AB51074) showing pro-, active, and degradation forms of MT1-MMP protein in MCF-7 MT1-MMP cell lines. β-actin was used as a loading control. c Gelatin zymography analysis of MCF-7 MT1-MMP cell lines incubated for 6 or 12 h with either MMP-2 CM alone, or in combination with rTIMP-2 at 100 ng/ml, or rTIMP-2 and BB94 (10 μm). Cells were also incubated for 12 h in MMP-2 CM supplemented with TIMP-2 CM diluted 1:100 in SF media ( bottom ). Bar graph shows densitometry quantification of MMP-2 isoforms from representative zymography of MCF-7 MT1-MMP cell lines incubated with MMP-2 CM and TIMP-2 CM diluted 1:100

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Stable Transfection, Zymography, Incubation

    MT1-MMP expression was inversely correlated to the extravasation efficiency of MCF-7 breast cancer cells in vivo. a Representative 3D volume views at 20× magnification of MCF-7 MT1-MMP cells stably expressing zsGreen 24 h-post intravenous injection into the chicken embryo CAM vasculature. Shown is an overlay displaying the zsGreen cells ( green ) and CAM vasculature and underlying stromal vessels labeled using lectin-rhodamine ( red ), and the isolated zsGreen channel. Scale bars = 100 μm. Bar graph shows quantification of extravasation efficiency of MCF-7 MT1-MMP cell lines 24 h post-injection. b Orthogonal views of Z-stacks acquired using confocal microscopy at 60× of MT1-MMP C1 and C3 cells 24 h post-injection showing the top of the CAM capillary bed ( top ) and the underlying stroma ( bottom ). Extravasated MT1-MMP C1 cells display loss of cell fragments ( green arrows ) and membrane blebbing ( white arrow ), whereas MT1-MMP C3 cells extravasate to below the CAM with uniform morphology ( blue arrows ) and are capable of forming invasive protrusions in the stroma ( red arrow ). Scale bars = 100 μm

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MT1-MMP expression was inversely correlated to the extravasation efficiency of MCF-7 breast cancer cells in vivo. a Representative 3D volume views at 20× magnification of MCF-7 MT1-MMP cells stably expressing zsGreen 24 h-post intravenous injection into the chicken embryo CAM vasculature. Shown is an overlay displaying the zsGreen cells ( green ) and CAM vasculature and underlying stromal vessels labeled using lectin-rhodamine ( red ), and the isolated zsGreen channel. Scale bars = 100 μm. Bar graph shows quantification of extravasation efficiency of MCF-7 MT1-MMP cell lines 24 h post-injection. b Orthogonal views of Z-stacks acquired using confocal microscopy at 60× of MT1-MMP C1 and C3 cells 24 h post-injection showing the top of the CAM capillary bed ( top ) and the underlying stroma ( bottom ). Extravasated MT1-MMP C1 cells display loss of cell fragments ( green arrows ) and membrane blebbing ( white arrow ), whereas MT1-MMP C3 cells extravasate to below the CAM with uniform morphology ( blue arrows ) and are capable of forming invasive protrusions in the stroma ( red arrow ). Scale bars = 100 μm

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Expressing, In Vivo, Stable Transfection, Injection, Chick Chorioallantoic Membrane Assay, Labeling, Isolation, Confocal Microscopy

    Low MT1-MMP/high TIMP-2 was optimal to promote migration and ERK activation in MCF-7 cells. a ERK activation in MCF-7 and MT1-MMP cells after incubation for 12 h (top) or 15 min ( bottom ) in media containing 10 % FBS or different dilutions of TIMP-2 or ALA + TIMP-2 CM in SF media. b Scratch closure migration assay of MCF-7 MT1-MMP cell lines monitored for 3 days. Shown are representative 10× fields of view. The white dotted lines indicate the initial scratch size; red dotted lines indicate the scratch size at the respective day. Scale bars = 100 μm. Line graph on the right shows scratch closure quantification that demonstrates significantly increased migratory potential of C3 cells. c Transwell migration assays of MCF-7 MT1-MMP cell lines incubated for 48 h in TIMP-2, or ALA + TIMP-2 CM diluted 1:100 ( top ), or ALA + TIMP-2 CM in increasing dilutions ( bottom ). d ( top ) qPCR analysis showing MT1-MMP mRNA from two cell lines derived from MT1-MMP C3 cells that stably express an shRNA construct targeting MT1-MMP, and one cell line stably expressing a control scrambled shRNA construct. Different letters indicate significant differences at p ≤ 0.05 by one-way ANOVA, Tukey’s post-hoc test. Individual student’s t-tests comparing MCF-7 cells against the C3 SH 1 cell line is also shown. ( bottom ) Transwell migration assay of MT1-MMP C3 cell lines incubated for 48 h in either TIMP-2 or ALA + TIMP-2 CM diluted 1:10, or ALA + TIMP2/U0126 (10 μm)

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: Low MT1-MMP/high TIMP-2 was optimal to promote migration and ERK activation in MCF-7 cells. a ERK activation in MCF-7 and MT1-MMP cells after incubation for 12 h (top) or 15 min ( bottom ) in media containing 10 % FBS or different dilutions of TIMP-2 or ALA + TIMP-2 CM in SF media. b Scratch closure migration assay of MCF-7 MT1-MMP cell lines monitored for 3 days. Shown are representative 10× fields of view. The white dotted lines indicate the initial scratch size; red dotted lines indicate the scratch size at the respective day. Scale bars = 100 μm. Line graph on the right shows scratch closure quantification that demonstrates significantly increased migratory potential of C3 cells. c Transwell migration assays of MCF-7 MT1-MMP cell lines incubated for 48 h in TIMP-2, or ALA + TIMP-2 CM diluted 1:100 ( top ), or ALA + TIMP-2 CM in increasing dilutions ( bottom ). d ( top ) qPCR analysis showing MT1-MMP mRNA from two cell lines derived from MT1-MMP C3 cells that stably express an shRNA construct targeting MT1-MMP, and one cell line stably expressing a control scrambled shRNA construct. Different letters indicate significant differences at p ≤ 0.05 by one-way ANOVA, Tukey’s post-hoc test. Individual student’s t-tests comparing MCF-7 cells against the C3 SH 1 cell line is also shown. ( bottom ) Transwell migration assay of MT1-MMP C3 cell lines incubated for 48 h in either TIMP-2 or ALA + TIMP-2 CM diluted 1:10, or ALA + TIMP2/U0126 (10 μm)

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Migration, Activation Assay, Incubation, Real-time Polymerase Chain Reaction, Derivative Assay, Stable Transfection, shRNA, Construct, Expressing, Transwell Migration Assay

    MCF-7 cell lines that express high levels of MT1-MMP demonstrated widespread ECM degradation (a) MCF-7, MT1-MMP cell lines, and MDA-MB 231 breast cancer cells were incubated on Alexa488 gelatin-coated coverslips for 24 h and processed for immunofluorescence to examine cytoplasmic MT1-MMP protein and ECM degradation. Representative fields of view are shown at 20× ( top panels ) and 60× ( bottom panels ) magnification. Panels are composed of an overlay showing the nuclei, F-actin, and Alexa488 gelatin signal ( top ) and the MT1-MMP signal with an inset of the Alexa488 gelatin channel ( bottom ). White arrows indicate cells that have degraded the underlying gelatin but are devoid of MT1-MMP signal. Scale bars = 100 μm. Cells in each sample positive for cytoplasmic MT1-MMP protein signal (MT1-MMP +) or devoid of underneath Alexa488 gelatin signal (Gelatin -) were quantified per 20× fields of view and are shown as mean percentage of total cells per field of view ± SEM

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MCF-7 cell lines that express high levels of MT1-MMP demonstrated widespread ECM degradation (a) MCF-7, MT1-MMP cell lines, and MDA-MB 231 breast cancer cells were incubated on Alexa488 gelatin-coated coverslips for 24 h and processed for immunofluorescence to examine cytoplasmic MT1-MMP protein and ECM degradation. Representative fields of view are shown at 20× ( top panels ) and 60× ( bottom panels ) magnification. Panels are composed of an overlay showing the nuclei, F-actin, and Alexa488 gelatin signal ( top ) and the MT1-MMP signal with an inset of the Alexa488 gelatin channel ( bottom ). White arrows indicate cells that have degraded the underlying gelatin but are devoid of MT1-MMP signal. Scale bars = 100 μm. Cells in each sample positive for cytoplasmic MT1-MMP protein signal (MT1-MMP +) or devoid of underneath Alexa488 gelatin signal (Gelatin -) were quantified per 20× fields of view and are shown as mean percentage of total cells per field of view ± SEM

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Multiple Displacement Amplification, Incubation, Immunofluorescence

    Kinase Activity Is Necessary for KIN10-Dependent WRI1 Degradation.

    Journal:

    Article Title: Phosphorylation of WRINKLED1 by KIN10 Results in Its Proteasomal Degradation, Providing a Link between Energy Homeostasis and Lipid Biosynthesis

    doi: 10.1105/tpc.17.00019

    Figure Lengend Snippet: Kinase Activity Is Necessary for KIN10-Dependent WRI1 Degradation.

    Article Snippet: Anti-WRI1 polyclonal antibodies were raised in rabbits immunized with a synthetic peptide (DFMFDDGKHECLNLENLDC) corresponding to residues 299 to 317 of the WRI1 amino acid sequence (Pierce, Thermo Fisher).

    Techniques: Activity Assay

    Localization of KIN10-Dependent WRI1 Phosphorylation Sites to Thr-70 and Ser-166 in Its AP2 DNA Binding Domains.

    Journal:

    Article Title: Phosphorylation of WRINKLED1 by KIN10 Results in Its Proteasomal Degradation, Providing a Link between Energy Homeostasis and Lipid Biosynthesis

    doi: 10.1105/tpc.17.00019

    Figure Lengend Snippet: Localization of KIN10-Dependent WRI1 Phosphorylation Sites to Thr-70 and Ser-166 in Its AP2 DNA Binding Domains.

    Article Snippet: Anti-WRI1 polyclonal antibodies were raised in rabbits immunized with a synthetic peptide (DFMFDDGKHECLNLENLDC) corresponding to residues 299 to 317 of the WRI1 amino acid sequence (Pierce, Thermo Fisher).

    Techniques: Binding Assay

    Phosphorylated WRI1 Can Be Detected in Extracts of N. benthamiana Leaves and B. napus Seeds.

    Journal:

    Article Title: Phosphorylation of WRINKLED1 by KIN10 Results in Its Proteasomal Degradation, Providing a Link between Energy Homeostasis and Lipid Biosynthesis

    doi: 10.1105/tpc.17.00019

    Figure Lengend Snippet: Phosphorylated WRI1 Can Be Detected in Extracts of N. benthamiana Leaves and B. napus Seeds.

    Article Snippet: Anti-WRI1 polyclonal antibodies were raised in rabbits immunized with a synthetic peptide (DFMFDDGKHECLNLENLDC) corresponding to residues 299 to 317 of the WRI1 amino acid sequence (Pierce, Thermo Fisher).

    Techniques:

    Overexpression of KIN10 Reduces WRI1 Levels in Siliques.

    Journal:

    Article Title: Phosphorylation of WRINKLED1 by KIN10 Results in Its Proteasomal Degradation, Providing a Link between Energy Homeostasis and Lipid Biosynthesis

    doi: 10.1105/tpc.17.00019

    Figure Lengend Snippet: Overexpression of KIN10 Reduces WRI1 Levels in Siliques.

    Article Snippet: Anti-WRI1 polyclonal antibodies were raised in rabbits immunized with a synthetic peptide (DFMFDDGKHECLNLENLDC) corresponding to residues 299 to 317 of the WRI1 amino acid sequence (Pierce, Thermo Fisher).

    Techniques: Over Expression

    WRI1 Is Represented by an Ensemble of Molecular Masses in Arabidopsis.

    Journal:

    Article Title: Phosphorylation of WRINKLED1 by KIN10 Results in Its Proteasomal Degradation, Providing a Link between Energy Homeostasis and Lipid Biosynthesis

    doi: 10.1105/tpc.17.00019

    Figure Lengend Snippet: WRI1 Is Represented by an Ensemble of Molecular Masses in Arabidopsis.

    Article Snippet: Anti-WRI1 polyclonal antibodies were raised in rabbits immunized with a synthetic peptide (DFMFDDGKHECLNLENLDC) corresponding to residues 299 to 317 of the WRI1 amino acid sequence (Pierce, Thermo Fisher).

    Techniques:

    Schematic Diagram of the WRI1 Predicted Protein Sequence Illustrating the Locations of Known Domains.

    Journal:

    Article Title: Phosphorylation of WRINKLED1 by KIN10 Results in Its Proteasomal Degradation, Providing a Link between Energy Homeostasis and Lipid Biosynthesis

    doi: 10.1105/tpc.17.00019

    Figure Lengend Snippet: Schematic Diagram of the WRI1 Predicted Protein Sequence Illustrating the Locations of Known Domains.

    Article Snippet: Anti-WRI1 polyclonal antibodies were raised in rabbits immunized with a synthetic peptide (DFMFDDGKHECLNLENLDC) corresponding to residues 299 to 317 of the WRI1 amino acid sequence (Pierce, Thermo Fisher).

    Techniques: Sequencing

    Overexpression of KIN10 Results in WRI1 Degradation in N. benthamiana Leaves.

    Journal:

    Article Title: Phosphorylation of WRINKLED1 by KIN10 Results in Its Proteasomal Degradation, Providing a Link between Energy Homeostasis and Lipid Biosynthesis

    doi: 10.1105/tpc.17.00019

    Figure Lengend Snippet: Overexpression of KIN10 Results in WRI1 Degradation in N. benthamiana Leaves.

    Article Snippet: Anti-WRI1 polyclonal antibodies were raised in rabbits immunized with a synthetic peptide (DFMFDDGKHECLNLENLDC) corresponding to residues 299 to 317 of the WRI1 amino acid sequence (Pierce, Thermo Fisher).

    Techniques: Over Expression

    Phosphorylated WRI1 Is Degraded Faster Than Native WRI1 in a Cell-Free Degradation Assay.

    Journal:

    Article Title: Phosphorylation of WRINKLED1 by KIN10 Results in Its Proteasomal Degradation, Providing a Link between Energy Homeostasis and Lipid Biosynthesis

    doi: 10.1105/tpc.17.00019

    Figure Lengend Snippet: Phosphorylated WRI1 Is Degraded Faster Than Native WRI1 in a Cell-Free Degradation Assay.

    Article Snippet: Anti-WRI1 polyclonal antibodies were raised in rabbits immunized with a synthetic peptide (DFMFDDGKHECLNLENLDC) corresponding to residues 299 to 317 of the WRI1 amino acid sequence (Pierce, Thermo Fisher).

    Techniques: Degradation Assay

    The Proteasome Inhibitor Bortezomib (PS-341) Reduces WRI1 Degradation in Both WRI1- Transformed and WRI1-KIN10 -Cotransformed N. benthamiana Leaves.

    Journal:

    Article Title: Phosphorylation of WRINKLED1 by KIN10 Results in Its Proteasomal Degradation, Providing a Link between Energy Homeostasis and Lipid Biosynthesis

    doi: 10.1105/tpc.17.00019

    Figure Lengend Snippet: The Proteasome Inhibitor Bortezomib (PS-341) Reduces WRI1 Degradation in Both WRI1- Transformed and WRI1-KIN10 -Cotransformed N. benthamiana Leaves.

    Article Snippet: Anti-WRI1 polyclonal antibodies were raised in rabbits immunized with a synthetic peptide (DFMFDDGKHECLNLENLDC) corresponding to residues 299 to 317 of the WRI1 amino acid sequence (Pierce, Thermo Fisher).

    Techniques: Transformation Assay

    Spectrum for Phosphorylated Peptide SRGV S KY Generated by Chymotrypsin Digestion of WRI1.

    Journal:

    Article Title: Phosphorylation of WRINKLED1 by KIN10 Results in Its Proteasomal Degradation, Providing a Link between Energy Homeostasis and Lipid Biosynthesis

    doi: 10.1105/tpc.17.00019

    Figure Lengend Snippet: Spectrum for Phosphorylated Peptide SRGV S KY Generated by Chymotrypsin Digestion of WRI1.

    Article Snippet: Anti-WRI1 polyclonal antibodies were raised in rabbits immunized with a synthetic peptide (DFMFDDGKHECLNLENLDC) corresponding to residues 299 to 317 of the WRI1 amino acid sequence (Pierce, Thermo Fisher).

    Techniques: Generated

    KIN10-Dependent Phosphorylation of WRI1.

    Journal:

    Article Title: Phosphorylation of WRINKLED1 by KIN10 Results in Its Proteasomal Degradation, Providing a Link between Energy Homeostasis and Lipid Biosynthesis

    doi: 10.1105/tpc.17.00019

    Figure Lengend Snippet: KIN10-Dependent Phosphorylation of WRI1.

    Article Snippet: Anti-WRI1 polyclonal antibodies were raised in rabbits immunized with a synthetic peptide (DFMFDDGKHECLNLENLDC) corresponding to residues 299 to 317 of the WRI1 amino acid sequence (Pierce, Thermo Fisher).

    Techniques:

    KIN10 and KIN11 Interact with WRI1.

    Journal:

    Article Title: Phosphorylation of WRINKLED1 by KIN10 Results in Its Proteasomal Degradation, Providing a Link between Energy Homeostasis and Lipid Biosynthesis

    doi: 10.1105/tpc.17.00019

    Figure Lengend Snippet: KIN10 and KIN11 Interact with WRI1.

    Article Snippet: Anti-WRI1 polyclonal antibodies were raised in rabbits immunized with a synthetic peptide (DFMFDDGKHECLNLENLDC) corresponding to residues 299 to 317 of the WRI1 amino acid sequence (Pierce, Thermo Fisher).

    Techniques:

    WRI1 Is Ubiquitinated in Arabidopsis.

    Journal:

    Article Title: Phosphorylation of WRINKLED1 by KIN10 Results in Its Proteasomal Degradation, Providing a Link between Energy Homeostasis and Lipid Biosynthesis

    doi: 10.1105/tpc.17.00019

    Figure Lengend Snippet: WRI1 Is Ubiquitinated in Arabidopsis.

    Article Snippet: Anti-WRI1 polyclonal antibodies were raised in rabbits immunized with a synthetic peptide (DFMFDDGKHECLNLENLDC) corresponding to residues 299 to 317 of the WRI1 amino acid sequence (Pierce, Thermo Fisher).

    Techniques:

    Lack of T-cadherin mRNA in the developing heart of mouse embryos at the E8.75–E10.5 stages ( 1 , 2 , 3 ). Arrows and the selected area indicate the developing heart region. Magnification of 5× ( 1 , 2 ) and 6× ( 3 )

    Journal: Acta Naturae

    Article Title: Detection of T-Cadherin Expression in Mouse Embryos

    doi:

    Figure Lengend Snippet: Lack of T-cadherin mRNA in the developing heart of mouse embryos at the E8.75–E10.5 stages ( 1 , 2 , 3 ). Arrows and the selected area indicate the developing heart region. Magnification of 5× ( 1 , 2 ) and 6× ( 3 )

    Article Snippet: T7 RNA polymerase (Promega) was used to synthesize the RNA probe for the T-cadherin sense sequence (negative control) from the linearized plasmid, and T3 RNA polymerase (Fermentas) was used to synthesize the antisense RNA probe for detection of the T-cadherin mRNA.

    Techniques:

    Immunofluorescent staining of mouse embryos at the E11.5 stage. Specific staining (red fluorescence) corresponds to expression of the T-cadherin protein. Blue fluorescence corresponds to nuclei additionally stained with DAPI. 1 – specific staining in the diencephalon region, as well as in the region of the developing eyecup; 2 – specific staining in the mesencephalon and metencephalon region; 3 – the same region as in 2 – at another optical plane level. Magnification of 5×

    Journal: Acta Naturae

    Article Title: Detection of T-Cadherin Expression in Mouse Embryos

    doi:

    Figure Lengend Snippet: Immunofluorescent staining of mouse embryos at the E11.5 stage. Specific staining (red fluorescence) corresponds to expression of the T-cadherin protein. Blue fluorescence corresponds to nuclei additionally stained with DAPI. 1 – specific staining in the diencephalon region, as well as in the region of the developing eyecup; 2 – specific staining in the mesencephalon and metencephalon region; 3 – the same region as in 2 – at another optical plane level. Magnification of 5×

    Article Snippet: T7 RNA polymerase (Promega) was used to synthesize the RNA probe for the T-cadherin sense sequence (negative control) from the linearized plasmid, and T3 RNA polymerase (Fermentas) was used to synthesize the antisense RNA probe for detection of the T-cadherin mRNA.

    Techniques: Staining, Fluorescence, Expressing

    In situ hybridization of mouse embryos at the of E8.75 stage. Expression of T-cadherin mRNA (T-cad): 1 – in the mesencephalon region; 2 – in the base of the developing optic vesicle; 3 – in the inner lining of the telencephalon; 4 – in the myelencephalon; 5 , 6 – in the optic vesicles. No specific staining in the negative control (control). Magnification of 3.2, 5, and 6×

    Journal: Acta Naturae

    Article Title: Detection of T-Cadherin Expression in Mouse Embryos

    doi:

    Figure Lengend Snippet: In situ hybridization of mouse embryos at the of E8.75 stage. Expression of T-cadherin mRNA (T-cad): 1 – in the mesencephalon region; 2 – in the base of the developing optic vesicle; 3 – in the inner lining of the telencephalon; 4 – in the myelencephalon; 5 , 6 – in the optic vesicles. No specific staining in the negative control (control). Magnification of 3.2, 5, and 6×

    Article Snippet: T7 RNA polymerase (Promega) was used to synthesize the RNA probe for the T-cadherin sense sequence (negative control) from the linearized plasmid, and T3 RNA polymerase (Fermentas) was used to synthesize the antisense RNA probe for detection of the T-cadherin mRNA.

    Techniques: In Situ, Hybridization, Expressing, Staining, Negative Control

    In situ hybridization of mouse embryos at the E9.5 stage. Staining of brain regions corresponds to the localization of T-cadherin mRNA (T-cad). Expression is observed in the base of the developing optic vesicles, in the parietal and occipital bend regions. Small arrows indicate the base of the developing optic vesicle. Control – the negative control. Magnification of 3.2, 5, and 6×

    Journal: Acta Naturae

    Article Title: Detection of T-Cadherin Expression in Mouse Embryos

    doi:

    Figure Lengend Snippet: In situ hybridization of mouse embryos at the E9.5 stage. Staining of brain regions corresponds to the localization of T-cadherin mRNA (T-cad). Expression is observed in the base of the developing optic vesicles, in the parietal and occipital bend regions. Small arrows indicate the base of the developing optic vesicle. Control – the negative control. Magnification of 3.2, 5, and 6×

    Article Snippet: T7 RNA polymerase (Promega) was used to synthesize the RNA probe for the T-cadherin sense sequence (negative control) from the linearized plasmid, and T3 RNA polymerase (Fermentas) was used to synthesize the antisense RNA probe for detection of the T-cadherin mRNA.

    Techniques: In Situ, Hybridization, Staining, Expressing, Negative Control

    In situ hybridization of mouse embryos at the E10.5 stage. Intense expression of T-cadherin (T-cad) in the developing tectum and the lateral regions of the diencephalon. The arrow indicates specific staining of the choroid plexus in the telencephalon region. No specific staining in the negative control (control). Magnification of 3.2, 5, and 6×

    Journal: Acta Naturae

    Article Title: Detection of T-Cadherin Expression in Mouse Embryos

    doi:

    Figure Lengend Snippet: In situ hybridization of mouse embryos at the E10.5 stage. Intense expression of T-cadherin (T-cad) in the developing tectum and the lateral regions of the diencephalon. The arrow indicates specific staining of the choroid plexus in the telencephalon region. No specific staining in the negative control (control). Magnification of 3.2, 5, and 6×

    Article Snippet: T7 RNA polymerase (Promega) was used to synthesize the RNA probe for the T-cadherin sense sequence (negative control) from the linearized plasmid, and T3 RNA polymerase (Fermentas) was used to synthesize the antisense RNA probe for detection of the T-cadherin mRNA.

    Techniques: In Situ, Hybridization, Expressing, Staining, Negative Control

    In situ hybridization of mouse embryos at the E10.5 stage. T-cad – specific staining of T-cadherin in the tectum (in the occipital bend region) and inner lining of the telencephalon; control – no specific staining in the negative control; Krox20 – staining of central nervous system structures in the positive control. Magnification of 3.2×

    Journal: Acta Naturae

    Article Title: Detection of T-Cadherin Expression in Mouse Embryos

    doi:

    Figure Lengend Snippet: In situ hybridization of mouse embryos at the E10.5 stage. T-cad – specific staining of T-cadherin in the tectum (in the occipital bend region) and inner lining of the telencephalon; control – no specific staining in the negative control; Krox20 – staining of central nervous system structures in the positive control. Magnification of 3.2×

    Article Snippet: T7 RNA polymerase (Promega) was used to synthesize the RNA probe for the T-cadherin sense sequence (negative control) from the linearized plasmid, and T3 RNA polymerase (Fermentas) was used to synthesize the antisense RNA probe for detection of the T-cadherin mRNA.

    Techniques: In Situ, Hybridization, Staining, Negative Control, Positive Control

    Immunofluorescent staining of mouse embryos at the E11.5 stage. Specific staining (red fluorescence) corresponds to expression of the T-cadherin protein (T-cad). Blue fluorescence corresponds to nuclei additionally stained with DAPI. 1 – specific staining reflecting T-cadherin expression in the heart region; 2 – control staining with antibodies to immunoglobulin G. Magnification of 5×

    Journal: Acta Naturae

    Article Title: Detection of T-Cadherin Expression in Mouse Embryos

    doi:

    Figure Lengend Snippet: Immunofluorescent staining of mouse embryos at the E11.5 stage. Specific staining (red fluorescence) corresponds to expression of the T-cadherin protein (T-cad). Blue fluorescence corresponds to nuclei additionally stained with DAPI. 1 – specific staining reflecting T-cadherin expression in the heart region; 2 – control staining with antibodies to immunoglobulin G. Magnification of 5×

    Article Snippet: T7 RNA polymerase (Promega) was used to synthesize the RNA probe for the T-cadherin sense sequence (negative control) from the linearized plasmid, and T3 RNA polymerase (Fermentas) was used to synthesize the antisense RNA probe for detection of the T-cadherin mRNA.

    Techniques: Staining, Fluorescence, Expressing

    Immunofluorescent staining of mouse embryos at the E9.5 (-1-) and E12.5 (-2-) stages. Specific staining (red fluorescence) corresponds to T-cadherin expression in the linings of the brain at both stages; expression of T-cadherin in the developing optic vesicle in the E9.5 embryo. Blue fluorescence corresponds to nuclei additionally stained with DAPI. Magnification of 5×

    Journal: Acta Naturae

    Article Title: Detection of T-Cadherin Expression in Mouse Embryos

    doi:

    Figure Lengend Snippet: Immunofluorescent staining of mouse embryos at the E9.5 (-1-) and E12.5 (-2-) stages. Specific staining (red fluorescence) corresponds to T-cadherin expression in the linings of the brain at both stages; expression of T-cadherin in the developing optic vesicle in the E9.5 embryo. Blue fluorescence corresponds to nuclei additionally stained with DAPI. Magnification of 5×

    Article Snippet: T7 RNA polymerase (Promega) was used to synthesize the RNA probe for the T-cadherin sense sequence (negative control) from the linearized plasmid, and T3 RNA polymerase (Fermentas) was used to synthesize the antisense RNA probe for detection of the T-cadherin mRNA.

    Techniques: Staining, Fluorescence, Expressing

    NPC2 deficiency confers fibroblast activation. A , expression of activated fibroblast markers as probed by real-time RT-PCR is shown. IL-6 and IL-1β secretion from non-elicited normal and NPC2-null human fibroblasts as measured by ELISA is shown.

    Journal:

    Article Title: Somatic Cell Plasticity and Niemann-Pick Type C2 Protein

    doi: 10.1074/jbc.M110.135897

    Figure Lengend Snippet: NPC2 deficiency confers fibroblast activation. A , expression of activated fibroblast markers as probed by real-time RT-PCR is shown. IL-6 and IL-1β secretion from non-elicited normal and NPC2-null human fibroblasts as measured by ELISA is shown.

    Article Snippet: The complimentary mRNA nucleotides derived from the human NPC2 sequence (Ambion, siRNA ID #18116) were transfected into human fibroblasts or primary human aortic SMC (Cambrex) plated onto a 96-well cell culture vessel using jetSI-ENDO Polyplus Transfection according to the manufacturer's instructions.

    Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Sustained ERK 1/2 activation in NPC2-null human fibroblasts. Total ( A ) and phosphorylated (activated, B ) ERK1/2 were detected by Western blotting in the cytosolic ( Cyt ) and nuclear protein ( N ) fractions isolated from the wild type ( WT ) and NPC2-null fibroblasts.

    Journal:

    Article Title: Somatic Cell Plasticity and Niemann-Pick Type C2 Protein

    doi: 10.1074/jbc.M110.135897

    Figure Lengend Snippet: Sustained ERK 1/2 activation in NPC2-null human fibroblasts. Total ( A ) and phosphorylated (activated, B ) ERK1/2 were detected by Western blotting in the cytosolic ( Cyt ) and nuclear protein ( N ) fractions isolated from the wild type ( WT ) and NPC2-null fibroblasts.

    Article Snippet: The complimentary mRNA nucleotides derived from the human NPC2 sequence (Ambion, siRNA ID #18116) were transfected into human fibroblasts or primary human aortic SMC (Cambrex) plated onto a 96-well cell culture vessel using jetSI-ENDO Polyplus Transfection according to the manufacturer's instructions.

    Techniques: Activation Assay, Western Blot, Isolation

    NPC2 deficiency results in enhanced cell migration; NPC2 null human dermal fibroblasts or normal human dermal fibroblasts after NPC2 gene silencing by siRNA and aortic SMC after NPC2 gene silencing by siRNA have increased migration toward 10 ng/ml PDGF-BB.

    Journal:

    Article Title: Somatic Cell Plasticity and Niemann-Pick Type C2 Protein

    doi: 10.1074/jbc.M110.135897

    Figure Lengend Snippet: NPC2 deficiency results in enhanced cell migration; NPC2 null human dermal fibroblasts or normal human dermal fibroblasts after NPC2 gene silencing by siRNA and aortic SMC after NPC2 gene silencing by siRNA have increased migration toward 10 ng/ml PDGF-BB.

    Article Snippet: The complimentary mRNA nucleotides derived from the human NPC2 sequence (Ambion, siRNA ID #18116) were transfected into human fibroblasts or primary human aortic SMC (Cambrex) plated onto a 96-well cell culture vessel using jetSI-ENDO Polyplus Transfection according to the manufacturer's instructions.

    Techniques: Migration

    Constitutive RTK/GFR activation in NPC2-null fibroblasts. The RTK/GFR activation was probed by human phospho-RTK antibody array using cytosolic fractions of cell lysates isolated from NPC2-null ( A ) and normal ( B ) human skin fibroblasts. Signals from particular

    Journal:

    Article Title: Somatic Cell Plasticity and Niemann-Pick Type C2 Protein

    doi: 10.1074/jbc.M110.135897

    Figure Lengend Snippet: Constitutive RTK/GFR activation in NPC2-null fibroblasts. The RTK/GFR activation was probed by human phospho-RTK antibody array using cytosolic fractions of cell lysates isolated from NPC2-null ( A ) and normal ( B ) human skin fibroblasts. Signals from particular

    Article Snippet: The complimentary mRNA nucleotides derived from the human NPC2 sequence (Ambion, siRNA ID #18116) were transfected into human fibroblasts or primary human aortic SMC (Cambrex) plated onto a 96-well cell culture vessel using jetSI-ENDO Polyplus Transfection according to the manufacturer's instructions.

    Techniques: Activation Assay, Ab Array, Isolation

    Scavenger receptor expression and function in NPC2-null human fibroblasts and aortic SMC. A , scavenger receptor expression and function in NPC2-null human fibroblasts compared with normal human fibroblasts is shown. B , scavenger receptor expression in

    Journal:

    Article Title: Somatic Cell Plasticity and Niemann-Pick Type C2 Protein

    doi: 10.1074/jbc.M110.135897

    Figure Lengend Snippet: Scavenger receptor expression and function in NPC2-null human fibroblasts and aortic SMC. A , scavenger receptor expression and function in NPC2-null human fibroblasts compared with normal human fibroblasts is shown. B , scavenger receptor expression in

    Article Snippet: The complimentary mRNA nucleotides derived from the human NPC2 sequence (Ambion, siRNA ID #18116) were transfected into human fibroblasts or primary human aortic SMC (Cambrex) plated onto a 96-well cell culture vessel using jetSI-ENDO Polyplus Transfection according to the manufacturer's instructions.

    Techniques: Expressing

    NPC2 Deficiency Confers Fibroblast Activation

    Journal:

    Article Title: Somatic Cell Plasticity and Niemann-Pick Type C2 Protein

    doi: 10.1074/jbc.M110.135897

    Figure Lengend Snippet: NPC2 Deficiency Confers Fibroblast Activation

    Article Snippet: The complimentary mRNA nucleotides derived from the human NPC2 sequence (Ambion, siRNA ID #18116) were transfected into human fibroblasts or primary human aortic SMC (Cambrex) plated onto a 96-well cell culture vessel using jetSI-ENDO Polyplus Transfection according to the manufacturer's instructions.

    Techniques: Activation Assay

    Cholesterol normalization in NPC2-null cells failed to correct their activated phenotype; real-time RT-PCR analysis of activated fibroblast markers ( A ) and scavenger receptors ( B ) in NPC2-null human skin fibroblasts. Cholesterol normalization ( Normal

    Journal:

    Article Title: Somatic Cell Plasticity and Niemann-Pick Type C2 Protein

    doi: 10.1074/jbc.M110.135897

    Figure Lengend Snippet: Cholesterol normalization in NPC2-null cells failed to correct their activated phenotype; real-time RT-PCR analysis of activated fibroblast markers ( A ) and scavenger receptors ( B ) in NPC2-null human skin fibroblasts. Cholesterol normalization ( Normal

    Article Snippet: The complimentary mRNA nucleotides derived from the human NPC2 sequence (Ambion, siRNA ID #18116) were transfected into human fibroblasts or primary human aortic SMC (Cambrex) plated onto a 96-well cell culture vessel using jetSI-ENDO Polyplus Transfection according to the manufacturer's instructions.

    Techniques: Quantitative RT-PCR

    Analysis of NPC2 gene expression in synovial fibroblasts isolated from patients with rheumatoid arthritis. The NPC2 mRNA transcript was measured by the real-time RT-PCR in five individual specimens designated A–E . Data normalized to the respective

    Journal:

    Article Title: Somatic Cell Plasticity and Niemann-Pick Type C2 Protein

    doi: 10.1074/jbc.M110.135897

    Figure Lengend Snippet: Analysis of NPC2 gene expression in synovial fibroblasts isolated from patients with rheumatoid arthritis. The NPC2 mRNA transcript was measured by the real-time RT-PCR in five individual specimens designated A–E . Data normalized to the respective

    Article Snippet: The complimentary mRNA nucleotides derived from the human NPC2 sequence (Ambion, siRNA ID #18116) were transfected into human fibroblasts or primary human aortic SMC (Cambrex) plated onto a 96-well cell culture vessel using jetSI-ENDO Polyplus Transfection according to the manufacturer's instructions.

    Techniques: Expressing, Isolation, Quantitative RT-PCR

    Cholesterol Accumulation Plays No Measurable Role in Either the Induction or the Maintenance of the Activated Fibroblast Phenotype in NPC2-null Cells

    Journal:

    Article Title: Somatic Cell Plasticity and Niemann-Pick Type C2 Protein

    doi: 10.1074/jbc.M110.135897

    Figure Lengend Snippet: Cholesterol Accumulation Plays No Measurable Role in Either the Induction or the Maintenance of the Activated Fibroblast Phenotype in NPC2-null Cells

    Article Snippet: The complimentary mRNA nucleotides derived from the human NPC2 sequence (Ambion, siRNA ID #18116) were transfected into human fibroblasts or primary human aortic SMC (Cambrex) plated onto a 96-well cell culture vessel using jetSI-ENDO Polyplus Transfection according to the manufacturer's instructions.

    Techniques:

    Lineage tracing of cells with Lgr5 transcriptional activity in H felis –infected Lgr5-Cre;H + /K + -Nog;Rosa26-Tom mice. 1-2-month old Lgr5-Cre;H + /K + -Nog;Rosa26-tdTom mice were treated with one i.p. injection of tamoxifen (0.1 mg/g body weight) and inoculated with H. felis two months post tamoxifen. Animals were analyzed 3 months after inoculation, and 5 months after tamoxifen injection. Gastric paraffin sections of the lesser curvature of H felis –infected Lgr5-Cre;H + /K + -Nog;Rosa26-tdTom mice were stained with anti-tomato (red fluorescent protein) primary antibodies and Alexa 555–conjugated secondary antibodies together with anti-IF antibodies and Alexa 488–conjugated secondary antibodies (IF/Tom), Alexa 488–conjugated GSII (GSII/Tom), Alexa 488–conjugated Ulex europaeus agglutinin 1 (UEA1), anti-CD44 and anti-CD44v9 primary antibodies and Alexa 488–conjugated secondary antibodies (CD44/Tom and CD44v 9/Tom), and anti-BrdU primary antibodies and Alexa 488–conjugated secondary antibodies (BrdU/Tom). Scale bars : 50 μm. Similar results were observed in at least 5 other H felis –infected Lgr5-Cre;H + /K + -Nog;Rosa26-tdTom mice except for the experiments with the anti-CD44v9 antibodies that were repeated in 1 other mouse.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Regulation of Gastric Lgr5+ve Cell Homeostasis by Bone Morphogenetic Protein (BMP) Signaling and Inflammatory Stimuli

    doi: 10.1016/j.jcmgh.2018.01.007

    Figure Lengend Snippet: Lineage tracing of cells with Lgr5 transcriptional activity in H felis –infected Lgr5-Cre;H + /K + -Nog;Rosa26-Tom mice. 1-2-month old Lgr5-Cre;H + /K + -Nog;Rosa26-tdTom mice were treated with one i.p. injection of tamoxifen (0.1 mg/g body weight) and inoculated with H. felis two months post tamoxifen. Animals were analyzed 3 months after inoculation, and 5 months after tamoxifen injection. Gastric paraffin sections of the lesser curvature of H felis –infected Lgr5-Cre;H + /K + -Nog;Rosa26-tdTom mice were stained with anti-tomato (red fluorescent protein) primary antibodies and Alexa 555–conjugated secondary antibodies together with anti-IF antibodies and Alexa 488–conjugated secondary antibodies (IF/Tom), Alexa 488–conjugated GSII (GSII/Tom), Alexa 488–conjugated Ulex europaeus agglutinin 1 (UEA1), anti-CD44 and anti-CD44v9 primary antibodies and Alexa 488–conjugated secondary antibodies (CD44/Tom and CD44v 9/Tom), and anti-BrdU primary antibodies and Alexa 488–conjugated secondary antibodies (BrdU/Tom). Scale bars : 50 μm. Similar results were observed in at least 5 other H felis –infected Lgr5-Cre;H + /K + -Nog;Rosa26-tdTom mice except for the experiments with the anti-CD44v9 antibodies that were repeated in 1 other mouse.

    Article Snippet: Pathogen-free C57BL/6 mice, Lgr5-enhanced green fluorescent protein (EGFP)-ires-CreERT2 (Lgr5-Cre ) mice, in which the expression of both Cre and green fluorescent protein (GFP ) is driven by endogenous Lgr5 regulatory sequences, and Rosa26-tdTom (Rosa26-Tom ) mice aged 4–10 weeks were purchased from Jackson Laboratory (Bar Harbor, ME).

    Techniques: Activity Assay, Infection, Mouse Assay, Injection, Staining

    Expression of IF in cells with Lgr5 transcriptional activity. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. Paraffin sections of the lesser curvature of Lgr5-Cre and of Lgr5-Cre;Bmpr1a flox-flox mice in the presence and absence of H felis (H. f.) were stained with anti-IF primary antibodies and an Alexa 555–conjugated secondary antibody (red) together with Alexa 488–conjugated anti-GFP antibodies (green). Scale bar : 25 μm. Similar results were observed in at least 1 other mouse and in 6 other mice in the H felis –infected Lgr5-Cre;Bmpr1a flox-flox group. DAPI, 4'6-diamidino-2-phenylindole.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Regulation of Gastric Lgr5+ve Cell Homeostasis by Bone Morphogenetic Protein (BMP) Signaling and Inflammatory Stimuli

    doi: 10.1016/j.jcmgh.2018.01.007

    Figure Lengend Snippet: Expression of IF in cells with Lgr5 transcriptional activity. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. Paraffin sections of the lesser curvature of Lgr5-Cre and of Lgr5-Cre;Bmpr1a flox-flox mice in the presence and absence of H felis (H. f.) were stained with anti-IF primary antibodies and an Alexa 555–conjugated secondary antibody (red) together with Alexa 488–conjugated anti-GFP antibodies (green). Scale bar : 25 μm. Similar results were observed in at least 1 other mouse and in 6 other mice in the H felis –infected Lgr5-Cre;Bmpr1a flox-flox group. DAPI, 4'6-diamidino-2-phenylindole.

    Article Snippet: Pathogen-free C57BL/6 mice, Lgr5-enhanced green fluorescent protein (EGFP)-ires-CreERT2 (Lgr5-Cre ) mice, in which the expression of both Cre and green fluorescent protein (GFP ) is driven by endogenous Lgr5 regulatory sequences, and Rosa26-tdTom (Rosa26-Tom ) mice aged 4–10 weeks were purchased from Jackson Laboratory (Bar Harbor, ME).

    Techniques: Expressing, Activity Assay, Mouse Assay, Injection, Staining, Infection

    Enhanced inflammatory changes, metaplasia, and dysplasia in the gastric epithelium of H felis –infected Lgr5-Cre;Bmpr1a flox-flox mice. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. ( A ) Representative H E-stained paraffin sections of the lesser curvature of Lgr5-Cre and of Lgr5-Cre;Bmpr1a flox-flox mice in the presence and absence of H felis (H. f.). The magnified window depicts dysplastic and inflammatory changes in H felis –infected Lgr5-Cre;Bmpr1a flox-flox mice. Scale bars : ( A ) 100 μm, ( B ) 25 μm. ( B ) Red arrows point to areas of dysplastic epithelium, black arrows indicate clusters of polymorphonuclear cells. ( C ) Graph bars represent the percentage of fields affected by gastritis, neutrophilic infiltrates, metaplasia, and dysplasia calculated in both Lgr5-Cre and Lgr5-Cre;Bmpr1a flox-flox mice in the presence and absence of H felis . Values are shown as means ± SE. Numbers in parenthesis indicate the number of animals used in each group. * P

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Regulation of Gastric Lgr5+ve Cell Homeostasis by Bone Morphogenetic Protein (BMP) Signaling and Inflammatory Stimuli

    doi: 10.1016/j.jcmgh.2018.01.007

    Figure Lengend Snippet: Enhanced inflammatory changes, metaplasia, and dysplasia in the gastric epithelium of H felis –infected Lgr5-Cre;Bmpr1a flox-flox mice. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. ( A ) Representative H E-stained paraffin sections of the lesser curvature of Lgr5-Cre and of Lgr5-Cre;Bmpr1a flox-flox mice in the presence and absence of H felis (H. f.). The magnified window depicts dysplastic and inflammatory changes in H felis –infected Lgr5-Cre;Bmpr1a flox-flox mice. Scale bars : ( A ) 100 μm, ( B ) 25 μm. ( B ) Red arrows point to areas of dysplastic epithelium, black arrows indicate clusters of polymorphonuclear cells. ( C ) Graph bars represent the percentage of fields affected by gastritis, neutrophilic infiltrates, metaplasia, and dysplasia calculated in both Lgr5-Cre and Lgr5-Cre;Bmpr1a flox-flox mice in the presence and absence of H felis . Values are shown as means ± SE. Numbers in parenthesis indicate the number of animals used in each group. * P

    Article Snippet: Pathogen-free C57BL/6 mice, Lgr5-enhanced green fluorescent protein (EGFP)-ires-CreERT2 (Lgr5-Cre ) mice, in which the expression of both Cre and green fluorescent protein (GFP ) is driven by endogenous Lgr5 regulatory sequences, and Rosa26-tdTom (Rosa26-Tom ) mice aged 4–10 weeks were purchased from Jackson Laboratory (Bar Harbor, ME).

    Techniques: Infection, Mouse Assay, Injection, Staining

    Expression of Bmpr1a , Id1 , and Lgr5 in the lesser and greater curvatures of Lgr5-Cre and Lgr5-Cre;Bmpr1a flox-flox mice. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and killed 5 months after tamoxifen. Bmpr1a , Id1 , and Lgr5 mRNA signals in the lesser and greater curvatures of Lgr5-Cre mice were compared with those detected in Lgr5-Cre;Bmpr1a flox-flox mice using QRT-PCR. Values are shown as means ± SE. Numbers in parenthesis indicate the number of animals used in each group. * P

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Regulation of Gastric Lgr5+ve Cell Homeostasis by Bone Morphogenetic Protein (BMP) Signaling and Inflammatory Stimuli

    doi: 10.1016/j.jcmgh.2018.01.007

    Figure Lengend Snippet: Expression of Bmpr1a , Id1 , and Lgr5 in the lesser and greater curvatures of Lgr5-Cre and Lgr5-Cre;Bmpr1a flox-flox mice. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and killed 5 months after tamoxifen. Bmpr1a , Id1 , and Lgr5 mRNA signals in the lesser and greater curvatures of Lgr5-Cre mice were compared with those detected in Lgr5-Cre;Bmpr1a flox-flox mice using QRT-PCR. Values are shown as means ± SE. Numbers in parenthesis indicate the number of animals used in each group. * P

    Article Snippet: Pathogen-free C57BL/6 mice, Lgr5-enhanced green fluorescent protein (EGFP)-ires-CreERT2 (Lgr5-Cre ) mice, in which the expression of both Cre and green fluorescent protein (GFP ) is driven by endogenous Lgr5 regulatory sequences, and Rosa26-tdTom (Rosa26-Tom ) mice aged 4–10 weeks were purchased from Jackson Laboratory (Bar Harbor, ME).

    Techniques: Expressing, Mouse Assay, Injection, Quantitative RT-PCR

    Distribution of tomato-positive cells in H felis –infected Lgr5-Cre;H + /K + -Nog;Rosa26-Tom mice. One- to 2-month-old Lgr5-Cre;Rosa26-Tom mice and Lgr5-Cre;H + /K + -Nog;Rosa26-tdTom mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. Gastric paraffin sections of the lesser curvature of Lgr5-Cre;Rosa26-Tom and Lgr5-Cre;H + /K + -Nog;Rosa26-tdTom in the presence and absence of H felis (H. f.) were stained with anti-tomato (red fluorescent protein) primary antibodies and Alexa 555–conjugated secondary antibodies. Matching sections of the gastric mucosa of both noninfected and H felis –infected Lgr5-Cre;H + /K + -Nog;Rosa26-tdTom mice were stained with H E. Similar results were observed in at least 3 other mice in each group. DAPI, 4′,6-diamidino-2-phenylindole.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Regulation of Gastric Lgr5+ve Cell Homeostasis by Bone Morphogenetic Protein (BMP) Signaling and Inflammatory Stimuli

    doi: 10.1016/j.jcmgh.2018.01.007

    Figure Lengend Snippet: Distribution of tomato-positive cells in H felis –infected Lgr5-Cre;H + /K + -Nog;Rosa26-Tom mice. One- to 2-month-old Lgr5-Cre;Rosa26-Tom mice and Lgr5-Cre;H + /K + -Nog;Rosa26-tdTom mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. Gastric paraffin sections of the lesser curvature of Lgr5-Cre;Rosa26-Tom and Lgr5-Cre;H + /K + -Nog;Rosa26-tdTom in the presence and absence of H felis (H. f.) were stained with anti-tomato (red fluorescent protein) primary antibodies and Alexa 555–conjugated secondary antibodies. Matching sections of the gastric mucosa of both noninfected and H felis –infected Lgr5-Cre;H + /K + -Nog;Rosa26-tdTom mice were stained with H E. Similar results were observed in at least 3 other mice in each group. DAPI, 4′,6-diamidino-2-phenylindole.

    Article Snippet: Pathogen-free C57BL/6 mice, Lgr5-enhanced green fluorescent protein (EGFP)-ires-CreERT2 (Lgr5-Cre ) mice, in which the expression of both Cre and green fluorescent protein (GFP ) is driven by endogenous Lgr5 regulatory sequences, and Rosa26-tdTom (Rosa26-Tom ) mice aged 4–10 weeks were purchased from Jackson Laboratory (Bar Harbor, ME).

    Techniques: Infection, Mouse Assay, Injection, Staining

    Expression of Cd44 and c-Myc mRNAs in the lesser curvature of Lgr5-Cre;Bmpr1a flox-flox mice. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. Cd44 and c-Myc mRNA signals in samples of the lesser curvature of Lgr5-Cre mice and of Lgr5-Cre;Bmpr1a flox-flox mice in the presence and absence of H felis (H. f.) were measured using QRT-PCR. Numbers in parenthesis indicate the number of animals used in each group. * P

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Regulation of Gastric Lgr5+ve Cell Homeostasis by Bone Morphogenetic Protein (BMP) Signaling and Inflammatory Stimuli

    doi: 10.1016/j.jcmgh.2018.01.007

    Figure Lengend Snippet: Expression of Cd44 and c-Myc mRNAs in the lesser curvature of Lgr5-Cre;Bmpr1a flox-flox mice. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. Cd44 and c-Myc mRNA signals in samples of the lesser curvature of Lgr5-Cre mice and of Lgr5-Cre;Bmpr1a flox-flox mice in the presence and absence of H felis (H. f.) were measured using QRT-PCR. Numbers in parenthesis indicate the number of animals used in each group. * P

    Article Snippet: Pathogen-free C57BL/6 mice, Lgr5-enhanced green fluorescent protein (EGFP)-ires-CreERT2 (Lgr5-Cre ) mice, in which the expression of both Cre and green fluorescent protein (GFP ) is driven by endogenous Lgr5 regulatory sequences, and Rosa26-tdTom (Rosa26-Tom ) mice aged 4–10 weeks were purchased from Jackson Laboratory (Bar Harbor, ME).

    Techniques: Expressing, Mouse Assay, Injection, Quantitative RT-PCR

    Development of SPEM in H felis –infected Lgr5-Cre;Bmpr1a flox-flox mice. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. Gastric paraffin sections of the lesser curvature of Lgr5-Cre and of Lgr5-Cre;Bmpr1a flox-flox mice in the presence and absence of H felis (H. f.) were stained with anti-IF primary antibodies and Alexa 555–conjugated secondary antibodies (red) together with Alexa 488–conjugated GS II (green). Gastric paraffin sections of the lesser curvature of H felis –infected Lgr5-Cre;Bmpr1a flox-flox mice were stained with anti-TFF2 primary antibodies and an Alexa 488–conjugated secondary antibody. Scale bar : 100 μm. Similar results were observed in at least 3 other mice in each group. DAPI, 4′,6-diamidino-2-phenylindole.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Regulation of Gastric Lgr5+ve Cell Homeostasis by Bone Morphogenetic Protein (BMP) Signaling and Inflammatory Stimuli

    doi: 10.1016/j.jcmgh.2018.01.007

    Figure Lengend Snippet: Development of SPEM in H felis –infected Lgr5-Cre;Bmpr1a flox-flox mice. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. Gastric paraffin sections of the lesser curvature of Lgr5-Cre and of Lgr5-Cre;Bmpr1a flox-flox mice in the presence and absence of H felis (H. f.) were stained with anti-IF primary antibodies and Alexa 555–conjugated secondary antibodies (red) together with Alexa 488–conjugated GS II (green). Gastric paraffin sections of the lesser curvature of H felis –infected Lgr5-Cre;Bmpr1a flox-flox mice were stained with anti-TFF2 primary antibodies and an Alexa 488–conjugated secondary antibody. Scale bar : 100 μm. Similar results were observed in at least 3 other mice in each group. DAPI, 4′,6-diamidino-2-phenylindole.

    Article Snippet: Pathogen-free C57BL/6 mice, Lgr5-enhanced green fluorescent protein (EGFP)-ires-CreERT2 (Lgr5-Cre ) mice, in which the expression of both Cre and green fluorescent protein (GFP ) is driven by endogenous Lgr5 regulatory sequences, and Rosa26-tdTom (Rosa26-Tom ) mice aged 4–10 weeks were purchased from Jackson Laboratory (Bar Harbor, ME).

    Techniques: Infection, Mouse Assay, Injection, Staining

    Expression of CD44 in H felis –infected Lgr5-Cre;Bmpr1a flox-flox mice. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. ( A ) Paraffin sections of the lesser curvature of Lgr5-Cre and Lgr5-Cre;Bmpr1a flox-flox mice in the presence and absence of H felis (H. f.) stained with anti-CD44 primary antibodies and a biotin-conjugated secondary antibody. The magnified window shows intense CD44 staining of epithelial cells in H felis –infected Lgr5-Cre;Bmpr1a flox-flox mice. Scale bar : 100 μm, 50 μm in the magnified window. Arrows point to CD44 +ve cells. ( B ) Paraffin sections of the lesser curvature of noninfected and H felis –infected Lgr5-Cre;Bmpr1a flox-flox mice stained with anti-CD44v9 primary antibodies and Alexa 488–conjugated secondary antibodies. The magnified window shows CD44v9 staining of epithelial cells at the base of glands in H felis –infected Lgr5-Cre;Bmpr1a flox-flox mice. Scale bar : 100 μm, 50 μm in the magnified window. Arrows point at CD44v9 +ve cells. ( C ) Paraffin sections of the lesser curvature of H felis –infected Lgr5-Cre;Bmpr1a flox-flox mice stained with anti-CD44 primary antibodies and Alexa 594-conjugated secondary antibodies ( red ) together with Alexa 488–conjugated GSII ( green ), anti-CD44 primary antibodies and Alexa 488–conjugated secondary antibodies ( green ) together with IF antibodies and Alexa 555–conjugated secondary antibodies ( red ) and anti-CD44 primary antibodies and Alexa 488–conjugated secondary antibodies ( green ) together with anti-Ki67 antibodies and Alexa 555–conjugated secondary antibodies ( red ). Similar results were observed in at least 2 other mice in each group. Scale bar : 50 μm. DAPI, 4′,6-diamidino-2-phenylindole.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Regulation of Gastric Lgr5+ve Cell Homeostasis by Bone Morphogenetic Protein (BMP) Signaling and Inflammatory Stimuli

    doi: 10.1016/j.jcmgh.2018.01.007

    Figure Lengend Snippet: Expression of CD44 in H felis –infected Lgr5-Cre;Bmpr1a flox-flox mice. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. ( A ) Paraffin sections of the lesser curvature of Lgr5-Cre and Lgr5-Cre;Bmpr1a flox-flox mice in the presence and absence of H felis (H. f.) stained with anti-CD44 primary antibodies and a biotin-conjugated secondary antibody. The magnified window shows intense CD44 staining of epithelial cells in H felis –infected Lgr5-Cre;Bmpr1a flox-flox mice. Scale bar : 100 μm, 50 μm in the magnified window. Arrows point to CD44 +ve cells. ( B ) Paraffin sections of the lesser curvature of noninfected and H felis –infected Lgr5-Cre;Bmpr1a flox-flox mice stained with anti-CD44v9 primary antibodies and Alexa 488–conjugated secondary antibodies. The magnified window shows CD44v9 staining of epithelial cells at the base of glands in H felis –infected Lgr5-Cre;Bmpr1a flox-flox mice. Scale bar : 100 μm, 50 μm in the magnified window. Arrows point at CD44v9 +ve cells. ( C ) Paraffin sections of the lesser curvature of H felis –infected Lgr5-Cre;Bmpr1a flox-flox mice stained with anti-CD44 primary antibodies and Alexa 594-conjugated secondary antibodies ( red ) together with Alexa 488–conjugated GSII ( green ), anti-CD44 primary antibodies and Alexa 488–conjugated secondary antibodies ( green ) together with IF antibodies and Alexa 555–conjugated secondary antibodies ( red ) and anti-CD44 primary antibodies and Alexa 488–conjugated secondary antibodies ( green ) together with anti-Ki67 antibodies and Alexa 555–conjugated secondary antibodies ( red ). Similar results were observed in at least 2 other mice in each group. Scale bar : 50 μm. DAPI, 4′,6-diamidino-2-phenylindole.

    Article Snippet: Pathogen-free C57BL/6 mice, Lgr5-enhanced green fluorescent protein (EGFP)-ires-CreERT2 (Lgr5-Cre ) mice, in which the expression of both Cre and green fluorescent protein (GFP ) is driven by endogenous Lgr5 regulatory sequences, and Rosa26-tdTom (Rosa26-Tom ) mice aged 4–10 weeks were purchased from Jackson Laboratory (Bar Harbor, ME).

    Techniques: Expressing, Infection, Mouse Assay, Injection, Staining

    Regulation of cells with Lgr5 transcriptional activity by BMP signaling. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis for 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. ( A ) Paraffin sections of the lesser and greater curvatures of Lgr5-Cre and Lgr5-Cre;Bmpr1a flox-flox mice were stained with Alexa 488–conjugated anti-GFP antibodies (green) together with an anti-H + ,K + -ATPase α-subunit primary antibody and an Alexa 555–conjugated secondary antibody (red). Scale bar : 50 μm. ( C ) Paraffin sections of the lesser curvature of Lgr5-Cre and Lgr5-Cre;Bmpr1a flox-flox mice were stained with Alexa 488–conjugated anti-GFP antibodies (green) together with an anti-Ki67 antibody and an Alexa 555–conjugated secondary antibody (red). Scale bar : 20 μm. Arrows point to GFP/Ki67-positive cells. Bars represent the number of ( B ) GFP- and of ( D ) GFP/Ki67-positive cells that were detected in the mucosa of the lesser curvature of Lgr5-Cre mice and of Lgr5-Cre;Bmpr1a flox-flox mice in the presence and absence of H felis (H. f.). Values are shown as means ± SE. Numbers in parenthesis indicate the number of animals used in each group. * P

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Regulation of Gastric Lgr5+ve Cell Homeostasis by Bone Morphogenetic Protein (BMP) Signaling and Inflammatory Stimuli

    doi: 10.1016/j.jcmgh.2018.01.007

    Figure Lengend Snippet: Regulation of cells with Lgr5 transcriptional activity by BMP signaling. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis for 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. ( A ) Paraffin sections of the lesser and greater curvatures of Lgr5-Cre and Lgr5-Cre;Bmpr1a flox-flox mice were stained with Alexa 488–conjugated anti-GFP antibodies (green) together with an anti-H + ,K + -ATPase α-subunit primary antibody and an Alexa 555–conjugated secondary antibody (red). Scale bar : 50 μm. ( C ) Paraffin sections of the lesser curvature of Lgr5-Cre and Lgr5-Cre;Bmpr1a flox-flox mice were stained with Alexa 488–conjugated anti-GFP antibodies (green) together with an anti-Ki67 antibody and an Alexa 555–conjugated secondary antibody (red). Scale bar : 20 μm. Arrows point to GFP/Ki67-positive cells. Bars represent the number of ( B ) GFP- and of ( D ) GFP/Ki67-positive cells that were detected in the mucosa of the lesser curvature of Lgr5-Cre mice and of Lgr5-Cre;Bmpr1a flox-flox mice in the presence and absence of H felis (H. f.). Values are shown as means ± SE. Numbers in parenthesis indicate the number of animals used in each group. * P

    Article Snippet: Pathogen-free C57BL/6 mice, Lgr5-enhanced green fluorescent protein (EGFP)-ires-CreERT2 (Lgr5-Cre ) mice, in which the expression of both Cre and green fluorescent protein (GFP ) is driven by endogenous Lgr5 regulatory sequences, and Rosa26-tdTom (Rosa26-Tom ) mice aged 4–10 weeks were purchased from Jackson Laboratory (Bar Harbor, ME).

    Techniques: Activity Assay, Mouse Assay, Injection, Staining

    Deletion of Bmpr1a in Lgr5 cells. GFP +ve cells from 1- to 2-month-old Lgr5-EGFP-ires-CreERT2 mice ( Lgr5-Cre mice) and Lgr5-EGFP-ires-CreERT2;Bmpr1a flox-flox mice ( Lgr5-Cre;Bmpr1a flox-flox mice) were isolated by flow cytometry. Mice were treated with 1 intraperitoneal. injection of tamoxifen (0.1 mg/g body weight) and killed 15 days after tamoxifen. Flow cytometry plots of cells isolated from ( A ) Lgr5-Cre mice and ( B ) Lgr5-Cre;Bmpr1a flox-flox mice. ( C ) Flow cytometry plot of cells isolated from nontransgenic negative control mice. ( D ) Bmpr1a mRNA signals in cells from Lgr5-Cre;Bmpr1a flox-flox mice were compared with those detected in cells from control mice using QRT-PCR. Values are shown as means ± SE. Numbers in parenthesis indicate the number of animals used in each group. * P

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Regulation of Gastric Lgr5+ve Cell Homeostasis by Bone Morphogenetic Protein (BMP) Signaling and Inflammatory Stimuli

    doi: 10.1016/j.jcmgh.2018.01.007

    Figure Lengend Snippet: Deletion of Bmpr1a in Lgr5 cells. GFP +ve cells from 1- to 2-month-old Lgr5-EGFP-ires-CreERT2 mice ( Lgr5-Cre mice) and Lgr5-EGFP-ires-CreERT2;Bmpr1a flox-flox mice ( Lgr5-Cre;Bmpr1a flox-flox mice) were isolated by flow cytometry. Mice were treated with 1 intraperitoneal. injection of tamoxifen (0.1 mg/g body weight) and killed 15 days after tamoxifen. Flow cytometry plots of cells isolated from ( A ) Lgr5-Cre mice and ( B ) Lgr5-Cre;Bmpr1a flox-flox mice. ( C ) Flow cytometry plot of cells isolated from nontransgenic negative control mice. ( D ) Bmpr1a mRNA signals in cells from Lgr5-Cre;Bmpr1a flox-flox mice were compared with those detected in cells from control mice using QRT-PCR. Values are shown as means ± SE. Numbers in parenthesis indicate the number of animals used in each group. * P

    Article Snippet: Pathogen-free C57BL/6 mice, Lgr5-enhanced green fluorescent protein (EGFP)-ires-CreERT2 (Lgr5-Cre ) mice, in which the expression of both Cre and green fluorescent protein (GFP ) is driven by endogenous Lgr5 regulatory sequences, and Rosa26-tdTom (Rosa26-Tom ) mice aged 4–10 weeks were purchased from Jackson Laboratory (Bar Harbor, ME).

    Techniques: Mouse Assay, Isolation, Flow Cytometry, Cytometry, Injection, Negative Control, Quantitative RT-PCR

    Decreased parietal cells and increased cell proliferation in Helicobacter -infected Lgr5-Cre;Bmpr1a flox-flox mice. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. Paraffin sections of the lesser curvature of Lgr5-Cre mice and of Lgr5-Cre;Bmpr1a flox-flox mice, in the presence and absence of H felis (H. f.), were stained with an ( A ) anti-H + ,K + -ATPase α-subunit primary antibody and an Alexa 488–conjugated secondary antibody (green), and with an ( C ) anti-Ki67 primary antibody and an Alexa 555–conjugated secondary antibody (red). Scale bars : 100 μm. Bars represent the number of ( B ) H + ,K + -ATPase α-subunit and of ( D ) Ki67-positive cells detected in Lgr5-Cre mice and in Lgr5-Cre;Bmpr1a flox-flox mice in the presence and absence of H felis . Values are shown as means ± SE. Numbers in parenthesis indicate the number of animals used in each group. * P

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Regulation of Gastric Lgr5+ve Cell Homeostasis by Bone Morphogenetic Protein (BMP) Signaling and Inflammatory Stimuli

    doi: 10.1016/j.jcmgh.2018.01.007

    Figure Lengend Snippet: Decreased parietal cells and increased cell proliferation in Helicobacter -infected Lgr5-Cre;Bmpr1a flox-flox mice. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. Paraffin sections of the lesser curvature of Lgr5-Cre mice and of Lgr5-Cre;Bmpr1a flox-flox mice, in the presence and absence of H felis (H. f.), were stained with an ( A ) anti-H + ,K + -ATPase α-subunit primary antibody and an Alexa 488–conjugated secondary antibody (green), and with an ( C ) anti-Ki67 primary antibody and an Alexa 555–conjugated secondary antibody (red). Scale bars : 100 μm. Bars represent the number of ( B ) H + ,K + -ATPase α-subunit and of ( D ) Ki67-positive cells detected in Lgr5-Cre mice and in Lgr5-Cre;Bmpr1a flox-flox mice in the presence and absence of H felis . Values are shown as means ± SE. Numbers in parenthesis indicate the number of animals used in each group. * P

    Article Snippet: Pathogen-free C57BL/6 mice, Lgr5-enhanced green fluorescent protein (EGFP)-ires-CreERT2 (Lgr5-Cre ) mice, in which the expression of both Cre and green fluorescent protein (GFP ) is driven by endogenous Lgr5 regulatory sequences, and Rosa26-tdTom (Rosa26-Tom ) mice aged 4–10 weeks were purchased from Jackson Laboratory (Bar Harbor, ME).

    Techniques: Infection, Mouse Assay, Injection, Staining

    SMPDL3A expression in cholesterol-loaded human monocyte-derived macrophages. A, SMPDL3A mRNA expression. HMDM from five individual healthy donors were incubated for 48 h in RPMI 1640 medium containing 10% (v/v) LPDS ± AcLDL (50 μg/ml),

    Journal:

    Article Title:

    doi: 10.1074/jbc.M114.612341

    Figure Lengend Snippet: SMPDL3A expression in cholesterol-loaded human monocyte-derived macrophages. A, SMPDL3A mRNA expression. HMDM from five individual healthy donors were incubated for 48 h in RPMI 1640 medium containing 10% (v/v) LPDS ± AcLDL (50 μg/ml),

    Article Snippet: A plasmid bearing the full-length sequence-verified human SMPDL3A open reading frame (IMAGE clone 100009862; Open Biosystems) was used as a template for all cloning experiments.

    Techniques: Expressing, Derivative Assay, Incubation

    SMPDL3A is secreted from HMDMs, N -glycosylated, and present in human serum. A, HMDM cell lysates and conditioned media from T-0901317-treated cells were incubated ± PNGaseF, separated by SDS-PAGE, and analyzed by Western blotting for human SMPDL3A

    Journal:

    Article Title:

    doi: 10.1074/jbc.M114.612341

    Figure Lengend Snippet: SMPDL3A is secreted from HMDMs, N -glycosylated, and present in human serum. A, HMDM cell lysates and conditioned media from T-0901317-treated cells were incubated ± PNGaseF, separated by SDS-PAGE, and analyzed by Western blotting for human SMPDL3A

    Article Snippet: A plasmid bearing the full-length sequence-verified human SMPDL3A open reading frame (IMAGE clone 100009862; Open Biosystems) was used as a template for all cloning experiments.

    Techniques: Incubation, SDS Page, Western Blot

    Secreted SMPDL3A levels and p-NP-TMP/ATP hydrolytic activity from LXR-agonist treated human macrophages. A, PMA differentiated THP-1 macrophages grown in RPMI 1640 medium, 10% FBS were transfected with 50 pmol of SMPDL3A siRNA or scrambled nonsilencing

    Journal:

    Article Title:

    doi: 10.1074/jbc.M114.612341

    Figure Lengend Snippet: Secreted SMPDL3A levels and p-NP-TMP/ATP hydrolytic activity from LXR-agonist treated human macrophages. A, PMA differentiated THP-1 macrophages grown in RPMI 1640 medium, 10% FBS were transfected with 50 pmol of SMPDL3A siRNA or scrambled nonsilencing

    Article Snippet: A plasmid bearing the full-length sequence-verified human SMPDL3A open reading frame (IMAGE clone 100009862; Open Biosystems) was used as a template for all cloning experiments.

    Techniques: Activity Assay, Transfection

    Expression, secretion, purification, glycosylation, and localization of recombinant human SMPDL3A in CHO cells. A, expression of human SMPDL3A in CHO-SMPDL3A cells was induced by addition of ± tetracycline (1 μ m , 16 h). Levels of SMPDL3A

    Journal:

    Article Title:

    doi: 10.1074/jbc.M114.612341

    Figure Lengend Snippet: Expression, secretion, purification, glycosylation, and localization of recombinant human SMPDL3A in CHO cells. A, expression of human SMPDL3A in CHO-SMPDL3A cells was induced by addition of ± tetracycline (1 μ m , 16 h). Levels of SMPDL3A

    Article Snippet: A plasmid bearing the full-length sequence-verified human SMPDL3A open reading frame (IMAGE clone 100009862; Open Biosystems) was used as a template for all cloning experiments.

    Techniques: Expressing, Purification, Recombinant

    SMPDL3A mRNA expression, protein expression, and secretion is regulated by cellular cAMP. A, SMPDL3A mRNA expression and comparison with SMPD1 . HMDM were incubated for 24 h in RPMI 1640 medium containing 10% FCS plus dibutyryl-cAMP ( db-cAMP , 1 m m ) and/or

    Journal:

    Article Title:

    doi: 10.1074/jbc.M114.612341

    Figure Lengend Snippet: SMPDL3A mRNA expression, protein expression, and secretion is regulated by cellular cAMP. A, SMPDL3A mRNA expression and comparison with SMPD1 . HMDM were incubated for 24 h in RPMI 1640 medium containing 10% FCS plus dibutyryl-cAMP ( db-cAMP , 1 m m ) and/or

    Article Snippet: A plasmid bearing the full-length sequence-verified human SMPDL3A open reading frame (IMAGE clone 100009862; Open Biosystems) was used as a template for all cloning experiments.

    Techniques: Expressing, Incubation

    Sphingomyelinase activity assays of recombinant human SMPDL3A. A, sphingomyelinase activity assay of lysates of CHO-SMPDL3A cells treated ± tetracycline (1 μ m , 16 h), using [ 3 H]choline methyl-labeled sphingomyelin/Triton X-100 micelles

    Journal:

    Article Title:

    doi: 10.1074/jbc.M114.612341

    Figure Lengend Snippet: Sphingomyelinase activity assays of recombinant human SMPDL3A. A, sphingomyelinase activity assay of lysates of CHO-SMPDL3A cells treated ± tetracycline (1 μ m , 16 h), using [ 3 H]choline methyl-labeled sphingomyelin/Triton X-100 micelles

    Article Snippet: A plasmid bearing the full-length sequence-verified human SMPDL3A open reading frame (IMAGE clone 100009862; Open Biosystems) was used as a template for all cloning experiments.

    Techniques: Activity Assay, Recombinant, Labeling

    A, nucleotide phosphodiesterase activity of recombinant SMPDL3A against nucleotide phosphate substrates. The activity of concentrated and buffer-exchanged conditioned media from CHO-SMPDL3A cells treated with or without 1 μ m tetracycline for 16

    Journal:

    Article Title:

    doi: 10.1074/jbc.M114.612341

    Figure Lengend Snippet: A, nucleotide phosphodiesterase activity of recombinant SMPDL3A against nucleotide phosphate substrates. The activity of concentrated and buffer-exchanged conditioned media from CHO-SMPDL3A cells treated with or without 1 μ m tetracycline for 16

    Article Snippet: A plasmid bearing the full-length sequence-verified human SMPDL3A open reading frame (IMAGE clone 100009862; Open Biosystems) was used as a template for all cloning experiments.

    Techniques: Activity Assay, Recombinant

    Activity of recombinant human SMPDL3A against synthetic p -nitrophenyl phosphodiester substrates. A, activity of conditioned serum-free media harvested from 1 × 10 6 control or tetracycline-induced CHO-SMPDL3A cells against p-NPP, bis-p-NPP, p-NPPC,

    Journal:

    Article Title:

    doi: 10.1074/jbc.M114.612341

    Figure Lengend Snippet: Activity of recombinant human SMPDL3A against synthetic p -nitrophenyl phosphodiester substrates. A, activity of conditioned serum-free media harvested from 1 × 10 6 control or tetracycline-induced CHO-SMPDL3A cells against p-NPP, bis-p-NPP, p-NPPC,

    Article Snippet: A plasmid bearing the full-length sequence-verified human SMPDL3A open reading frame (IMAGE clone 100009862; Open Biosystems) was used as a template for all cloning experiments.

    Techniques: Activity Assay, Recombinant

    Amino acid sequence comparison of SMPDL3A with acid sphingomyelinase and SMPDL3B. The amino acid sequences of human SMPDL3A and close human homologs, SMPDL3B and aSMase (encoded by the SMPD1 gene), were aligned using ClustalW2. Asterisks indicate exact

    Journal:

    Article Title:

    doi: 10.1074/jbc.M114.612341

    Figure Lengend Snippet: Amino acid sequence comparison of SMPDL3A with acid sphingomyelinase and SMPDL3B. The amino acid sequences of human SMPDL3A and close human homologs, SMPDL3B and aSMase (encoded by the SMPD1 gene), were aligned using ClustalW2. Asterisks indicate exact

    Article Snippet: A plasmid bearing the full-length sequence-verified human SMPDL3A open reading frame (IMAGE clone 100009862; Open Biosystems) was used as a template for all cloning experiments.

    Techniques: Sequencing

    Identification of contraction-independent FSHD patients carrying deletions of an intronic FAT1 enhancer. ( A ) View of the Human genomic FAT1 locus focusing on an area including FAT1 exons 17-18-19. The lower image is a USCC browser based screen-copy image showing a track displaying ENCODE enhancer and promoter associated histone mark (H3K4me1) on 8 cell lines. ( B ) Positions of copy number variants identified in 5 FSHD patients by CGH and positioned on the genome by CGHweb analysis. Patients are identified with a specific number, and their characteristics are available in the Table S1 . The deletion span varies from deletions restricted to intron 17 to deletions spanning over intron 17, exon 17 and intron 16, including a ENCODE-putative enhancer visible through genomic browsers. ( C ) Copy number validation of the deletion by qPCR. The three graphs show the relative amounts of PCR fragments obtained using primers couples 1–2 (exon 17), 2–3 (enhancer intron 16) and 4–5 (exon 16)), in a group of 40 healthy controls (blue area), a group of 10 FSHD1 patients (red area), and a group of 19 contraction-independent patients (c.i.FSHD). All data were normalized using an unrelated genomic fragment (Adora) as internal control, and one of the control DNAs (number 21) was used as the reference DNA (where all values are set to 1). A cut-off of 0.75 has been set. Individuals in which the relative value is lower than the cut-off are considered as having lowered copy numbers (indicated as loss). Information on each patient (regarding clinical and genetic diagnostic) are available in the Table S1 . ( D ) The distribution of CNVs corresponding to loss CNV (seen as red) is shown in controls and in FSHD groups (all together, or FSHD1 and c.i.FSHD separately) for each of the three spots considered individually (top three graphs) or considered together (bottom plot, where loss represents the number of cases having a loss for at least one of the three spots). The cases where a significant link (as measured by X 2 or Fischer tests) are indicated with one or two stars (* for p

    Journal: PLoS Genetics

    Article Title: Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy

    doi: 10.1371/journal.pgen.1003550

    Figure Lengend Snippet: Identification of contraction-independent FSHD patients carrying deletions of an intronic FAT1 enhancer. ( A ) View of the Human genomic FAT1 locus focusing on an area including FAT1 exons 17-18-19. The lower image is a USCC browser based screen-copy image showing a track displaying ENCODE enhancer and promoter associated histone mark (H3K4me1) on 8 cell lines. ( B ) Positions of copy number variants identified in 5 FSHD patients by CGH and positioned on the genome by CGHweb analysis. Patients are identified with a specific number, and their characteristics are available in the Table S1 . The deletion span varies from deletions restricted to intron 17 to deletions spanning over intron 17, exon 17 and intron 16, including a ENCODE-putative enhancer visible through genomic browsers. ( C ) Copy number validation of the deletion by qPCR. The three graphs show the relative amounts of PCR fragments obtained using primers couples 1–2 (exon 17), 2–3 (enhancer intron 16) and 4–5 (exon 16)), in a group of 40 healthy controls (blue area), a group of 10 FSHD1 patients (red area), and a group of 19 contraction-independent patients (c.i.FSHD). All data were normalized using an unrelated genomic fragment (Adora) as internal control, and one of the control DNAs (number 21) was used as the reference DNA (where all values are set to 1). A cut-off of 0.75 has been set. Individuals in which the relative value is lower than the cut-off are considered as having lowered copy numbers (indicated as loss). Information on each patient (regarding clinical and genetic diagnostic) are available in the Table S1 . ( D ) The distribution of CNVs corresponding to loss CNV (seen as red) is shown in controls and in FSHD groups (all together, or FSHD1 and c.i.FSHD separately) for each of the three spots considered individually (top three graphs) or considered together (bottom plot, where loss represents the number of cases having a loss for at least one of the three spots). The cases where a significant link (as measured by X 2 or Fischer tests) are indicated with one or two stars (* for p

    Article Snippet: Expression of the human FAT1 gene was monitored by a real time quantitative RT-PCR method using TaqMan gene expression assay reference number Hs00170627_m1 targeting the 5′ part of the FAT1 sequence (Applied biosystem), or using real-time sybrgreen PCR assay (Roche) (see primers below).

    Techniques: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Diagnostic Assay

    Presymptomatic adult Fat1 mutant mice show selective defects in scapular muscles. ( A ) Adult Fat1 LacZ/LacZ mice show visible scapular winging (orange arrow) at stages prior to detectable weight loss (defined as presymptomatic). Pictures (extracted from movies) show a posture in which the mice challenge their shoulder girdle muscles by extending their head as far rostral as possible. At 7 weeks, wasting of the rhomboid muscles can already be detected in presymptomatic Fat1 LacZ/LacZ mice as they move on a cage grid. Note the large gap (orange arrow) between scapulas (where rhomboids normally maintain scapulas attached to the dorsal spine), not visible in the corresponding position in the wild type littermate. ( B ) At advanced symptomatic stages (30% weight loss, anesthetized mice), there is marked curvature of the spine in the upper back and shoulder area, also visible through X-ray post-mortem imaging. ( C ) Kaplan-Meier plot showing survival of wild type, Fat1 LacZ/+ , and Fat1 LacZ/LacZ mice. Most Fat1 LacZ/LacZ mice die between 2 and 4 months, with a median survival of 3 months, while a small group survives beyond 6 months. ( D ) Masses of dissected muscles of Fat1 LacZ/LacZ mice at presymptomatic disease stage (0% weight loss, n = 3) relative to age-matched controls (n = 6; average wild type weight defined as 100%). ( E ) Motor performance defects in presymptomatic adult Fat1 LacZ/LacZ mice. Rotarod analysis shows that the latency to fall off from the rod was significantly shorter in presymptomatic adult Fat1 LacZ/LacZ . In this set of experiments, additional Fat1 LacZ/LacZ mice that were symptomatic at the stage when training started had died by the time the test was performed and are therefore not included in the graph. ( F ) Scapular muscle dissection in adult wild type and Fat1 LacZ/LacZ mice reveals a pronounced reduction in volume and thickness of the rhomboid superficialis (Rh. Sup.) and rhomboid profundus (Rb. P.). This likely underlies the scapular winging phenotype. In the top pictures, the trapezius cervicalis (Trap) has been removed on the right side of each mouse to uncover the other scapular muscles ( rhomboids : Rho; levator scapula : LS). Yellow dotted lines indicate the extent of the scapula, red and orange dotted lines that of the two rhomboid muscles. The intermediate magnification highlights the respective shapes of the rhomboid superficialis (orange dotted line) and rhomboid profundus (purple dotted line). ( G ) Phalloidin staining of flat-mounted rhomboid superficialis muscles of wild type and Fat1 LacZ/LacZ mice at presymptomatic (middle panel) or advanced disease (20% weight loss; bottom panel) stages shows that early defects of myofiber orientation precede reduction of myofibre diameter. Scale bars: ( F ) 2 mm; ( G ) 300 µm.

    Journal: PLoS Genetics

    Article Title: Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy

    doi: 10.1371/journal.pgen.1003550

    Figure Lengend Snippet: Presymptomatic adult Fat1 mutant mice show selective defects in scapular muscles. ( A ) Adult Fat1 LacZ/LacZ mice show visible scapular winging (orange arrow) at stages prior to detectable weight loss (defined as presymptomatic). Pictures (extracted from movies) show a posture in which the mice challenge their shoulder girdle muscles by extending their head as far rostral as possible. At 7 weeks, wasting of the rhomboid muscles can already be detected in presymptomatic Fat1 LacZ/LacZ mice as they move on a cage grid. Note the large gap (orange arrow) between scapulas (where rhomboids normally maintain scapulas attached to the dorsal spine), not visible in the corresponding position in the wild type littermate. ( B ) At advanced symptomatic stages (30% weight loss, anesthetized mice), there is marked curvature of the spine in the upper back and shoulder area, also visible through X-ray post-mortem imaging. ( C ) Kaplan-Meier plot showing survival of wild type, Fat1 LacZ/+ , and Fat1 LacZ/LacZ mice. Most Fat1 LacZ/LacZ mice die between 2 and 4 months, with a median survival of 3 months, while a small group survives beyond 6 months. ( D ) Masses of dissected muscles of Fat1 LacZ/LacZ mice at presymptomatic disease stage (0% weight loss, n = 3) relative to age-matched controls (n = 6; average wild type weight defined as 100%). ( E ) Motor performance defects in presymptomatic adult Fat1 LacZ/LacZ mice. Rotarod analysis shows that the latency to fall off from the rod was significantly shorter in presymptomatic adult Fat1 LacZ/LacZ . In this set of experiments, additional Fat1 LacZ/LacZ mice that were symptomatic at the stage when training started had died by the time the test was performed and are therefore not included in the graph. ( F ) Scapular muscle dissection in adult wild type and Fat1 LacZ/LacZ mice reveals a pronounced reduction in volume and thickness of the rhomboid superficialis (Rh. Sup.) and rhomboid profundus (Rb. P.). This likely underlies the scapular winging phenotype. In the top pictures, the trapezius cervicalis (Trap) has been removed on the right side of each mouse to uncover the other scapular muscles ( rhomboids : Rho; levator scapula : LS). Yellow dotted lines indicate the extent of the scapula, red and orange dotted lines that of the two rhomboid muscles. The intermediate magnification highlights the respective shapes of the rhomboid superficialis (orange dotted line) and rhomboid profundus (purple dotted line). ( G ) Phalloidin staining of flat-mounted rhomboid superficialis muscles of wild type and Fat1 LacZ/LacZ mice at presymptomatic (middle panel) or advanced disease (20% weight loss; bottom panel) stages shows that early defects of myofiber orientation precede reduction of myofibre diameter. Scale bars: ( F ) 2 mm; ( G ) 300 µm.

    Article Snippet: Expression of the human FAT1 gene was monitored by a real time quantitative RT-PCR method using TaqMan gene expression assay reference number Hs00170627_m1 targeting the 5′ part of the FAT1 sequence (Applied biosystem), or using real-time sybrgreen PCR assay (Roche) (see primers below).

    Techniques: Mutagenesis, Mouse Assay, Imaging, Dissection, Staining

    FAT1 protein and RNA levels are mis-regulated in human foetal FSHD tissues. ( A ) Immunolocalization of FAT1 (Rb-1465 anti FAT1-ICD, green) and DHPR (Cacna1s, magenta) in longitudinal sections from human quadriceps biopsies from a control (top) or and FSHD (F1, bottom) foetus with 1.5 D4Z4 repeats. ( B ) qPCR analysis of FAT1 mRNA levels in quadriceps (3 left graphs) and deltoid muscles (middle graph) and in brain (right graph), comparing respectively with age-matched control foetuses (blue bars), a 26 weeks old FSHD1 foetus (F1) harbouring 1.5 D4Z4 repeats in the 4q35 region (dark red bars), a 16 weeks old FSHD1 foetus harbouring 4.3 D4Z4 repeats at 4q35 region (F2), and twin FSHD1 foetuses aged 28 weeks, with 7 D4Z4 repeats. ( C ) Analysis of the regulatory status of the promoter region by Chromatin immunoprecipitation. The respective level of the following histone marks: H3K27me3 (silenced chromatin; C-left ), and H3K4m3 (promoter active; C-right ), in muscle extracts from four age matched controls (ct1 to 4) or four FSHD1 foetuses (F1 to F4) are shown. Relative quantities were normalized with the level of histone marks at the promoter of the GUSB gene as internal control, and expressed as % of control 1 (ct1). Scale bars: ( A ) 50 µm.

    Journal: PLoS Genetics

    Article Title: Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy

    doi: 10.1371/journal.pgen.1003550

    Figure Lengend Snippet: FAT1 protein and RNA levels are mis-regulated in human foetal FSHD tissues. ( A ) Immunolocalization of FAT1 (Rb-1465 anti FAT1-ICD, green) and DHPR (Cacna1s, magenta) in longitudinal sections from human quadriceps biopsies from a control (top) or and FSHD (F1, bottom) foetus with 1.5 D4Z4 repeats. ( B ) qPCR analysis of FAT1 mRNA levels in quadriceps (3 left graphs) and deltoid muscles (middle graph) and in brain (right graph), comparing respectively with age-matched control foetuses (blue bars), a 26 weeks old FSHD1 foetus (F1) harbouring 1.5 D4Z4 repeats in the 4q35 region (dark red bars), a 16 weeks old FSHD1 foetus harbouring 4.3 D4Z4 repeats at 4q35 region (F2), and twin FSHD1 foetuses aged 28 weeks, with 7 D4Z4 repeats. ( C ) Analysis of the regulatory status of the promoter region by Chromatin immunoprecipitation. The respective level of the following histone marks: H3K27me3 (silenced chromatin; C-left ), and H3K4m3 (promoter active; C-right ), in muscle extracts from four age matched controls (ct1 to 4) or four FSHD1 foetuses (F1 to F4) are shown. Relative quantities were normalized with the level of histone marks at the promoter of the GUSB gene as internal control, and expressed as % of control 1 (ct1). Scale bars: ( A ) 50 µm.

    Article Snippet: Expression of the human FAT1 gene was monitored by a real time quantitative RT-PCR method using TaqMan gene expression assay reference number Hs00170627_m1 targeting the 5′ part of the FAT1 sequence (Applied biosystem), or using real-time sybrgreen PCR assay (Roche) (see primers below).

    Techniques: Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

    Selective changes in Fat1 mutant mice recapitulate the clinical picture of FSHD. ( A ) Schematic representation of the human 4q35.2 region, including 5 Mb upstream of the FSHD-associated D4Z4 repeat array. ( B–C ) Retinal defects and exudative vasculopathy in adult Fat1 LacZ/LacZ retinas. Fat1 LacZ/LacZ eyes have an opaque appearance, in contrast to wild type eyes ( B ; yellow arrow). Removal of the cornea reveals absence of opening of the pigmented retina (aniridia), which therefore covers the lens and prevents light from entering the eye. ( C ) Retinal vasculature visualized using isolectinB4 (GS-IB4) staining of flat-mounted adult retinas from wild type and Fat1 LacZ/LacZ mice. The retina of Fat1 LacZ/LacZ mice displayed zones in which the normal net of secondary and tertiary vessels was replaced by disorganized vasculature, revealing numerous intra-retinal microvascular abnormalities, including IB4-binding microaneurysms (orange arrows). Insert: Example of severe retinal detachment (red arrows) observed in Fat1 LacZ/LacZ eyes, visible even through the lens prior to its removal during dissection. ( D ) The shape of the inner ear was visualized at E12.5 in WT and Fat1 ΔTM/ΔTM embryos owing to the MLC3F-2E transgene, which is expressed in the developing inner ear in addition to differentiating muscles. Micrographs show an area of the face around the ear. This area shows: left: the masserter muscles (unaffected), bottom: a stream of muscle cells migrating subcutaneously from the second brachial arch (future subcutaneous muscles of the face, which migration path is visibly affected); and top right: the inner ear structure with the endolymphatic duct (ed), a long tube oriented dorsally, finishing with an enlarged area called the endolymphatic sac (es). Both the ed and es are reduced in half Fat1 ΔTM/ΔTM inner ears examined (frequently asymmetric). ( E ) Quantification of the inner ear shape defect was performed by measuring the area occupied by the endolymphatic duct (ed) and endolymphatic sac (es), as illustrated with the red dotted lines in ( D ). Each value for a given genotype were plotted on a vertical line, to illustrate the scale of variability of mutant phenotypes. Scale bars: ( B,C 3 ) 0,5 mm; ( C 1–2 ) 200 µm; ( C 4–5 ) 80 µm, ( C 6 ) 30 µm.

    Journal: PLoS Genetics

    Article Title: Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy

    doi: 10.1371/journal.pgen.1003550

    Figure Lengend Snippet: Selective changes in Fat1 mutant mice recapitulate the clinical picture of FSHD. ( A ) Schematic representation of the human 4q35.2 region, including 5 Mb upstream of the FSHD-associated D4Z4 repeat array. ( B–C ) Retinal defects and exudative vasculopathy in adult Fat1 LacZ/LacZ retinas. Fat1 LacZ/LacZ eyes have an opaque appearance, in contrast to wild type eyes ( B ; yellow arrow). Removal of the cornea reveals absence of opening of the pigmented retina (aniridia), which therefore covers the lens and prevents light from entering the eye. ( C ) Retinal vasculature visualized using isolectinB4 (GS-IB4) staining of flat-mounted adult retinas from wild type and Fat1 LacZ/LacZ mice. The retina of Fat1 LacZ/LacZ mice displayed zones in which the normal net of secondary and tertiary vessels was replaced by disorganized vasculature, revealing numerous intra-retinal microvascular abnormalities, including IB4-binding microaneurysms (orange arrows). Insert: Example of severe retinal detachment (red arrows) observed in Fat1 LacZ/LacZ eyes, visible even through the lens prior to its removal during dissection. ( D ) The shape of the inner ear was visualized at E12.5 in WT and Fat1 ΔTM/ΔTM embryos owing to the MLC3F-2E transgene, which is expressed in the developing inner ear in addition to differentiating muscles. Micrographs show an area of the face around the ear. This area shows: left: the masserter muscles (unaffected), bottom: a stream of muscle cells migrating subcutaneously from the second brachial arch (future subcutaneous muscles of the face, which migration path is visibly affected); and top right: the inner ear structure with the endolymphatic duct (ed), a long tube oriented dorsally, finishing with an enlarged area called the endolymphatic sac (es). Both the ed and es are reduced in half Fat1 ΔTM/ΔTM inner ears examined (frequently asymmetric). ( E ) Quantification of the inner ear shape defect was performed by measuring the area occupied by the endolymphatic duct (ed) and endolymphatic sac (es), as illustrated with the red dotted lines in ( D ). Each value for a given genotype were plotted on a vertical line, to illustrate the scale of variability of mutant phenotypes. Scale bars: ( B,C 3 ) 0,5 mm; ( C 1–2 ) 200 µm; ( C 4–5 ) 80 µm, ( C 6 ) 30 µm.

    Article Snippet: Expression of the human FAT1 gene was monitored by a real time quantitative RT-PCR method using TaqMan gene expression assay reference number Hs00170627_m1 targeting the 5′ part of the FAT1 sequence (Applied biosystem), or using real-time sybrgreen PCR assay (Roche) (see primers below).

    Techniques: Mutagenesis, Mouse Assay, Staining, Binding Assay, Dissection, Migration

    Fat1 expression at late stages of muscle differentiation. ( A ) Fat1 expression was visualized in E13.5 embryos or in neonate (P0) muscle by β-galactosidase staining or by in situ hybridization with a Fat1 3′UTR RNA probe. ( B – D ) Immunolocalization of FAT1 (anti-FAT1-ICD from [35] , green) was performed in E12.5 mouse embryo ( C ), and on adult ( B, D ) muscle fibers on longitudinal muscle cryosections from wild type ( B , C 1–3 , D 1,4 ), from Fat1 ΔTM/ΔTM embryos ( C 4–6 ), and from Fat1 LacZ/LacZ ( D 2–3 , D 5–6 ) mice, combined with either antibodies against alpha-actinin (red, B 5 ), DHPR (Cacna1s) (red, B 2,3 ), or RyR (red, B 4 ), or with Phalloidin (red, C, D ). In D , Green channel images (FAT1) were first captured with either identical exposure time between wild type and mutants ( D 1,4 and D 2,5 , 421 ms), or with longer exposure time ( D 3,6 , 2222 ms). This indicates that the epitope detected by the anti-FAT1-ICD antibody (from ref [35] ) is present in reduced but detectable amounts in Fat1 LacZ/LacZ muscles. This observation was made when Fat1 LacZ/LacZ mice (n = 2 at P0; and n = 3 at adult stages) displayed severe muscle defects at the stage of dissection, indicating that levels of FAT1 protein inversely correlate with phenotype severity. Scale bars: ( B – D ) 4 µm, ( C ) 6 µm.

    Journal: PLoS Genetics

    Article Title: Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy

    doi: 10.1371/journal.pgen.1003550

    Figure Lengend Snippet: Fat1 expression at late stages of muscle differentiation. ( A ) Fat1 expression was visualized in E13.5 embryos or in neonate (P0) muscle by β-galactosidase staining or by in situ hybridization with a Fat1 3′UTR RNA probe. ( B – D ) Immunolocalization of FAT1 (anti-FAT1-ICD from [35] , green) was performed in E12.5 mouse embryo ( C ), and on adult ( B, D ) muscle fibers on longitudinal muscle cryosections from wild type ( B , C 1–3 , D 1,4 ), from Fat1 ΔTM/ΔTM embryos ( C 4–6 ), and from Fat1 LacZ/LacZ ( D 2–3 , D 5–6 ) mice, combined with either antibodies against alpha-actinin (red, B 5 ), DHPR (Cacna1s) (red, B 2,3 ), or RyR (red, B 4 ), or with Phalloidin (red, C, D ). In D , Green channel images (FAT1) were first captured with either identical exposure time between wild type and mutants ( D 1,4 and D 2,5 , 421 ms), or with longer exposure time ( D 3,6 , 2222 ms). This indicates that the epitope detected by the anti-FAT1-ICD antibody (from ref [35] ) is present in reduced but detectable amounts in Fat1 LacZ/LacZ muscles. This observation was made when Fat1 LacZ/LacZ mice (n = 2 at P0; and n = 3 at adult stages) displayed severe muscle defects at the stage of dissection, indicating that levels of FAT1 protein inversely correlate with phenotype severity. Scale bars: ( B – D ) 4 µm, ( C ) 6 µm.

    Article Snippet: Expression of the human FAT1 gene was monitored by a real time quantitative RT-PCR method using TaqMan gene expression assay reference number Hs00170627_m1 targeting the 5′ part of the FAT1 sequence (Applied biosystem), or using real-time sybrgreen PCR assay (Roche) (see primers below).

    Techniques: Expressing, Staining, In Situ, Hybridization, Mouse Assay, Mass Spectrometry, Dissection

    Abnormally shaped shoulder muscles of Fat1 -deficient mice develop phenotypes involving reduced muscle fibres diameter and structural abnormalities. ( A ) Muscle architecture visualized on transverse ( A 1,2 ) or longitudinal ( A 3–6 ) sections of rhomboid muscles from wild type and Fat1 LacZ/LacZ mice (20% weight loss), using antibodies against laminin and α-actinin, or toluidine blue staining. ( B ) NMJs were visualized by immunolabeling nerve endings with anti-neurofilaments antibodies (NF, red) and AchR clusters with α-bungarotoxin (green). ( C ) Plot of muscle fiber diameter in scapular muscles ( rhomboid , trapezius , latissimus dorsi , and cutaneous maximus ) of adult Fat1 LacZ/LacZ mice at early symptomatic (n = 5, red bars) and advance stages (n = 7, green bars), compared to wild type littermates (n = 12, blue bars). ( D ) Electron micrographs at three different magnifications in rhomboid muscle fibres from Fat1 LacZ/LacZ adult mice at early symptomatic stages (6–15% weight loss) show fragmentation of the myofibre architecture and loss of t-tubule integrity. In wild type myofibres, t-tubules (purple arrows) are visible between myofibrils, precisely aligned on either side of each Z-band, at a position coinciding with the end of the myosin filaments. By contrast, in dystrophic fibres from Fat1 LacZ/LacZ mice, the general disorganization correlated with missing (stars), mis-oriented, mis-aligned (orange arrows), or fragmented (red arrows) triads. An increased distance (indicated as blue double arrowed bar) between the sarcolemma and contractile apparatus is observed in Fat1 LacZ/LacZ muscles, compared to wild types, indicating a loss of the tight association between the contractile apparatus and the sarcolemma. Scale bars: ( A 1–2 ) 50 µm; ( A 3–6 ) 20 µm; ( B ) 15 µm; ( D 1,2 ) 5 µm; ( D 3,4 ) 0.5 µm; ( D 5,6 ) 0.2 µm.

    Journal: PLoS Genetics

    Article Title: Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy

    doi: 10.1371/journal.pgen.1003550

    Figure Lengend Snippet: Abnormally shaped shoulder muscles of Fat1 -deficient mice develop phenotypes involving reduced muscle fibres diameter and structural abnormalities. ( A ) Muscle architecture visualized on transverse ( A 1,2 ) or longitudinal ( A 3–6 ) sections of rhomboid muscles from wild type and Fat1 LacZ/LacZ mice (20% weight loss), using antibodies against laminin and α-actinin, or toluidine blue staining. ( B ) NMJs were visualized by immunolabeling nerve endings with anti-neurofilaments antibodies (NF, red) and AchR clusters with α-bungarotoxin (green). ( C ) Plot of muscle fiber diameter in scapular muscles ( rhomboid , trapezius , latissimus dorsi , and cutaneous maximus ) of adult Fat1 LacZ/LacZ mice at early symptomatic (n = 5, red bars) and advance stages (n = 7, green bars), compared to wild type littermates (n = 12, blue bars). ( D ) Electron micrographs at three different magnifications in rhomboid muscle fibres from Fat1 LacZ/LacZ adult mice at early symptomatic stages (6–15% weight loss) show fragmentation of the myofibre architecture and loss of t-tubule integrity. In wild type myofibres, t-tubules (purple arrows) are visible between myofibrils, precisely aligned on either side of each Z-band, at a position coinciding with the end of the myosin filaments. By contrast, in dystrophic fibres from Fat1 LacZ/LacZ mice, the general disorganization correlated with missing (stars), mis-oriented, mis-aligned (orange arrows), or fragmented (red arrows) triads. An increased distance (indicated as blue double arrowed bar) between the sarcolemma and contractile apparatus is observed in Fat1 LacZ/LacZ muscles, compared to wild types, indicating a loss of the tight association between the contractile apparatus and the sarcolemma. Scale bars: ( A 1–2 ) 50 µm; ( A 3–6 ) 20 µm; ( B ) 15 µm; ( D 1,2 ) 5 µm; ( D 3,4 ) 0.5 µm; ( D 5,6 ) 0.2 µm.

    Article Snippet: Expression of the human FAT1 gene was monitored by a real time quantitative RT-PCR method using TaqMan gene expression assay reference number Hs00170627_m1 targeting the 5′ part of the FAT1 sequence (Applied biosystem), or using real-time sybrgreen PCR assay (Roche) (see primers below).

    Techniques: Mouse Assay, Staining, Immunolabeling

    The transmembrane domain of FAT1 is required to polarize muscle migration. ( A ) Schemes representing the main protein product expected from a wild type, a Fat1 LacZ , and a Fat1 ΔTM locus. Positions of the epitopes for three antibodies are also shown, with a color code matching that used in the western blots below. ( B ) Western blot analysis of the FAT1 protein products observed in total lysates from E12.5 Fat1 LacZ/LacZ , wild type, and Fat1 ΔTM/ΔTM embryos using indicated antibodies, which targeted epitopes are positioned in ( A ). ( C ) Whole mount LacZ staining of E12.5 Fat1 LacZ/LacZ mutant embryo. ( D ) Skeletal muscle groups were visualized in E12.5, E13.5, and E18.5 control and Fat1 ΔTM/ΔTM embryos carrying the MLC3f-2E transgene, by X-gal staining. Whole mount analysis of skeletal muscles confirms the presence of a reduced CM (red dotted lines) at E12.5, leading to a misshaped CM one day later (E13.5), and the systematic presence of ectopic muscles in the shoulder area (yellow arrow), most frequently inserting between the deltoid and triceps muscles. Flat mounted preparations of the CM dissected from an E18.5 Fat1 ΔTM/ΔTM embryo, showing the reduced density as well as randomly oriented multinucleated myofibres (right panels). ( E ) Whole mount in situ hybridization on E10.5 embryos with an RNA probe matching the Floxed exons (exons 24–25, the probe is indicated in yellow in Figure S4A ). The profile of Fat1 RNA expression in a wild type embryo matches previously reported expression domain, including staining in the limb, somites, branchial arches, telencephalon, midbrain, eye, tail bud, and neural tube roof plate. Fat1 ΔTM/ΔTM embryos are entirely devoid of staining, apart from the otic vesicle, a known site of substrate trapping (yielding background staining). In contrast, varying amounts of residual RNA were consistently observed in Fat1 LacZ/LacZ embryos, in the telencephalon, midbrain, limbs, tailbud, and somites. Two examples are shown with different RNA levels detected.

    Journal: PLoS Genetics

    Article Title: Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy

    doi: 10.1371/journal.pgen.1003550

    Figure Lengend Snippet: The transmembrane domain of FAT1 is required to polarize muscle migration. ( A ) Schemes representing the main protein product expected from a wild type, a Fat1 LacZ , and a Fat1 ΔTM locus. Positions of the epitopes for three antibodies are also shown, with a color code matching that used in the western blots below. ( B ) Western blot analysis of the FAT1 protein products observed in total lysates from E12.5 Fat1 LacZ/LacZ , wild type, and Fat1 ΔTM/ΔTM embryos using indicated antibodies, which targeted epitopes are positioned in ( A ). ( C ) Whole mount LacZ staining of E12.5 Fat1 LacZ/LacZ mutant embryo. ( D ) Skeletal muscle groups were visualized in E12.5, E13.5, and E18.5 control and Fat1 ΔTM/ΔTM embryos carrying the MLC3f-2E transgene, by X-gal staining. Whole mount analysis of skeletal muscles confirms the presence of a reduced CM (red dotted lines) at E12.5, leading to a misshaped CM one day later (E13.5), and the systematic presence of ectopic muscles in the shoulder area (yellow arrow), most frequently inserting between the deltoid and triceps muscles. Flat mounted preparations of the CM dissected from an E18.5 Fat1 ΔTM/ΔTM embryo, showing the reduced density as well as randomly oriented multinucleated myofibres (right panels). ( E ) Whole mount in situ hybridization on E10.5 embryos with an RNA probe matching the Floxed exons (exons 24–25, the probe is indicated in yellow in Figure S4A ). The profile of Fat1 RNA expression in a wild type embryo matches previously reported expression domain, including staining in the limb, somites, branchial arches, telencephalon, midbrain, eye, tail bud, and neural tube roof plate. Fat1 ΔTM/ΔTM embryos are entirely devoid of staining, apart from the otic vesicle, a known site of substrate trapping (yielding background staining). In contrast, varying amounts of residual RNA were consistently observed in Fat1 LacZ/LacZ embryos, in the telencephalon, midbrain, limbs, tailbud, and somites. Two examples are shown with different RNA levels detected.

    Article Snippet: Expression of the human FAT1 gene was monitored by a real time quantitative RT-PCR method using TaqMan gene expression assay reference number Hs00170627_m1 targeting the 5′ part of the FAT1 sequence (Applied biosystem), or using real-time sybrgreen PCR assay (Roche) (see primers below).

    Techniques: Migration, Western Blot, Staining, Mutagenesis, In Situ, Hybridization, RNA Expression, Expressing

    Fat1 controls the shape of subsets of scapular muscle by modulating myoblast polarity during planar migration. ( A–C ) Reporter gene expression in the forelimb and flank of mouse embryos between E11.5 and E13.5. ( A ) Gdnf-lacZ staining labels myoblasts of the latissimus dorsee (LD) and cutaneous maximus (CM). CM myoblasts migrate away from the brachial plexus to form a subcutaneous muscle sheath, composed of radially-oriented chains of myoblasts. ( B ) At E13.5, MLC3f-lacZ staining reveals the characteristic fan-shaped form of the CM (dotted white purple line) as compared to other limb muscles. ( C ) Fat1 expression detected using the lacZ gene trap allele KST249 ( Fat1 LacZ ) is selectively localized within the CM and in surrounding tissue (pink arrow). ( D ) CM myoblasts express Fat1 and migrate towards an increasing gradient of Fat1 expression. Alternate vibratome cross-sections of a wild type E12.5 embryo were hybridized with Fat1 (left column) and MyoD (purple, right column) RNA probes. Photographs of adjacent sections were superimposed (photoshop) after conversion of Fat1 staining color in pink (right column; Fat1 in pink, MyoD in purple). MyoD expression is used as a marker of the muscle lineage. Superimposition was meant to compare the relative levels of Fat1 expression within and around the cutaneous maximus (CM) muscle (indicated with purple arrows), at three consecutive antero-posterior positions, respectively within the CM (top row), at the posterior end (middle row), and posterior to the caudal extremity of the CM at that stage. CM myoblasts, migrating from anterior to posterior, express lower levels of Fat1 RNA than the surrounding subcutaneous cell layer (pink arrows). Intensity of Fat1 staining in this subcutaneous layer increases gradually in caudal sections. ( E–H ) Orientation of CM myoblast migration in whole-mounts of E12.5 Fat1 LacZ/LacZ and control embryos detected using MyoD in situ hybridization. In all panels anterior is to the left, dorsal is to the top. ( E ) The CM muscle (purple dotted line) in Fat1 LacZ/LacZ embryos displays reduced size and altered shape as compared to wild type. Higher magnification images (right hand panels) show that within the CM muscle, radial organization of myoblast chains was perturbed by Fat1 -deficiency, resulting in a fuzzy migration front and irregular distribution of myoblasts (red arrows). In addition, ectopic clusters of myoblasts (orange arrows) are detected in the shoulder area (dotted orange line). ( F ) Quantification of the abnormal orientation of Fat1 mutant myoblasts. The angle between the longest diameter of each myoblast nucleus and the axis of the closest myoblast chain was measured on flat-mounted CM muscles. The bar graph presents mean (± s.e.m.) percentages of myoD + nuclei displaying a given angle (by angle ranges of 10°) for wild type (gray) and Fat1 LacZ/LacZ (black) embryos. ( G, H ) High magnification images of MyoD -expressing myoblasts in equivalent positions – within the chains ( G ) or at the leading edge (migration front, H ) – in the CM of mutants and controls. Scale bars: ( A–C ), 0.8 mm; ( D ) 300 µm; ( E ), left: 0.5 mm; ( E ), right: 100 µm; ( G, H ) 10 µm.

    Journal: PLoS Genetics

    Article Title: Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy

    doi: 10.1371/journal.pgen.1003550

    Figure Lengend Snippet: Fat1 controls the shape of subsets of scapular muscle by modulating myoblast polarity during planar migration. ( A–C ) Reporter gene expression in the forelimb and flank of mouse embryos between E11.5 and E13.5. ( A ) Gdnf-lacZ staining labels myoblasts of the latissimus dorsee (LD) and cutaneous maximus (CM). CM myoblasts migrate away from the brachial plexus to form a subcutaneous muscle sheath, composed of radially-oriented chains of myoblasts. ( B ) At E13.5, MLC3f-lacZ staining reveals the characteristic fan-shaped form of the CM (dotted white purple line) as compared to other limb muscles. ( C ) Fat1 expression detected using the lacZ gene trap allele KST249 ( Fat1 LacZ ) is selectively localized within the CM and in surrounding tissue (pink arrow). ( D ) CM myoblasts express Fat1 and migrate towards an increasing gradient of Fat1 expression. Alternate vibratome cross-sections of a wild type E12.5 embryo were hybridized with Fat1 (left column) and MyoD (purple, right column) RNA probes. Photographs of adjacent sections were superimposed (photoshop) after conversion of Fat1 staining color in pink (right column; Fat1 in pink, MyoD in purple). MyoD expression is used as a marker of the muscle lineage. Superimposition was meant to compare the relative levels of Fat1 expression within and around the cutaneous maximus (CM) muscle (indicated with purple arrows), at three consecutive antero-posterior positions, respectively within the CM (top row), at the posterior end (middle row), and posterior to the caudal extremity of the CM at that stage. CM myoblasts, migrating from anterior to posterior, express lower levels of Fat1 RNA than the surrounding subcutaneous cell layer (pink arrows). Intensity of Fat1 staining in this subcutaneous layer increases gradually in caudal sections. ( E–H ) Orientation of CM myoblast migration in whole-mounts of E12.5 Fat1 LacZ/LacZ and control embryos detected using MyoD in situ hybridization. In all panels anterior is to the left, dorsal is to the top. ( E ) The CM muscle (purple dotted line) in Fat1 LacZ/LacZ embryos displays reduced size and altered shape as compared to wild type. Higher magnification images (right hand panels) show that within the CM muscle, radial organization of myoblast chains was perturbed by Fat1 -deficiency, resulting in a fuzzy migration front and irregular distribution of myoblasts (red arrows). In addition, ectopic clusters of myoblasts (orange arrows) are detected in the shoulder area (dotted orange line). ( F ) Quantification of the abnormal orientation of Fat1 mutant myoblasts. The angle between the longest diameter of each myoblast nucleus and the axis of the closest myoblast chain was measured on flat-mounted CM muscles. The bar graph presents mean (± s.e.m.) percentages of myoD + nuclei displaying a given angle (by angle ranges of 10°) for wild type (gray) and Fat1 LacZ/LacZ (black) embryos. ( G, H ) High magnification images of MyoD -expressing myoblasts in equivalent positions – within the chains ( G ) or at the leading edge (migration front, H ) – in the CM of mutants and controls. Scale bars: ( A–C ), 0.8 mm; ( D ) 300 µm; ( E ), left: 0.5 mm; ( E ), right: 100 µm; ( G, H ) 10 µm.

    Article Snippet: Expression of the human FAT1 gene was monitored by a real time quantitative RT-PCR method using TaqMan gene expression assay reference number Hs00170627_m1 targeting the 5′ part of the FAT1 sequence (Applied biosystem), or using real-time sybrgreen PCR assay (Roche) (see primers below).

    Techniques: Migration, Expressing, Staining, Marker, In Situ, Hybridization, Mutagenesis

    Ablation of Fat1 in premigratory myoblasts using Pax3-cre partially reproduces the muscle migration/shape abnormalities of the constitutive knockout. ( A ) Skeletal muscle cells were visualized at E12.5 in WT, Fat1 ΔTM/ΔTM , Fat1 Fln/Fln , and Fat1 Fln/Fln ; Pax3 cre/+ embryos, owing to the MLC3F-2E transgene by performing X-gal staining, after clearing in 100% glycerol. The upper panels show micrographs of the forelimb area, and indicate the positions at which higher magnification pictures shown in the two lower panels were taken. ( B, C ) The phenotype was quantified in WT, Fat1 ΔTM/ΔTM , Fat1 Fln/Fln , and Fat1 Fln/Fln ; Pax3 cre/+ as well as in the control genotypes in Fat1 ΔTM/+ , Fat1 Fln/+ and Fat1 Fln/+ ; Pax3 cre/+ and Pax3 cre/+ in two different manners: ( B ) by counting the number of dispersed myocytes found in the elbow area (orange dotted lines in the lower panels in ( A )), ( C ) by measuring the area occupied by the ectopically positioned muscle (or myocyte cluster) that appears inserted between (red dotted line in middle panels). All data from a given genotype are plotted on a vertical line. Overlapping dots were arbitrarily moved away from the vertical lines to allow showing all results distinctly. In both cases, the Fat1 ΔTM/ΔTM , Fat1 Fln/Fln , and Fat1 Fln/Fln ; Pax3 cre/+ groups were each significantly different from the control genotypes (WT, Fat1 Fln/+ , and Fat1 Fln/+ ; Pax3 cre/+ respectively, t-test, p values indicated), and were significantly different from each other ( Fat1 ΔTM/ΔTM from Fat1 Fln/Fln , and from Fat1 Fln/Fln ; Pax3 cre/+ , but also Fat1 Fln/Fln from Fat1 Fln/Fln ; Pax3 cre/+ , t-test, p values indicated).

    Journal: PLoS Genetics

    Article Title: Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy

    doi: 10.1371/journal.pgen.1003550

    Figure Lengend Snippet: Ablation of Fat1 in premigratory myoblasts using Pax3-cre partially reproduces the muscle migration/shape abnormalities of the constitutive knockout. ( A ) Skeletal muscle cells were visualized at E12.5 in WT, Fat1 ΔTM/ΔTM , Fat1 Fln/Fln , and Fat1 Fln/Fln ; Pax3 cre/+ embryos, owing to the MLC3F-2E transgene by performing X-gal staining, after clearing in 100% glycerol. The upper panels show micrographs of the forelimb area, and indicate the positions at which higher magnification pictures shown in the two lower panels were taken. ( B, C ) The phenotype was quantified in WT, Fat1 ΔTM/ΔTM , Fat1 Fln/Fln , and Fat1 Fln/Fln ; Pax3 cre/+ as well as in the control genotypes in Fat1 ΔTM/+ , Fat1 Fln/+ and Fat1 Fln/+ ; Pax3 cre/+ and Pax3 cre/+ in two different manners: ( B ) by counting the number of dispersed myocytes found in the elbow area (orange dotted lines in the lower panels in ( A )), ( C ) by measuring the area occupied by the ectopically positioned muscle (or myocyte cluster) that appears inserted between (red dotted line in middle panels). All data from a given genotype are plotted on a vertical line. Overlapping dots were arbitrarily moved away from the vertical lines to allow showing all results distinctly. In both cases, the Fat1 ΔTM/ΔTM , Fat1 Fln/Fln , and Fat1 Fln/Fln ; Pax3 cre/+ groups were each significantly different from the control genotypes (WT, Fat1 Fln/+ , and Fat1 Fln/+ ; Pax3 cre/+ respectively, t-test, p values indicated), and were significantly different from each other ( Fat1 ΔTM/ΔTM from Fat1 Fln/Fln , and from Fat1 Fln/Fln ; Pax3 cre/+ , but also Fat1 Fln/Fln from Fat1 Fln/Fln ; Pax3 cre/+ , t-test, p values indicated).

    Article Snippet: Expression of the human FAT1 gene was monitored by a real time quantitative RT-PCR method using TaqMan gene expression assay reference number Hs00170627_m1 targeting the 5′ part of the FAT1 sequence (Applied biosystem), or using real-time sybrgreen PCR assay (Roche) (see primers below).

    Techniques: Migration, Knock-Out, Staining

    Fat1 loss of function alters shapes of selective facial and scapulohumeral muscles. Skeletal muscle groups were visualized in E14.5, E15.5, and E18.5 wild type and Fat1 ΔTM/ΔTM embryos carrying the MLC3f-2E (LacZ) transgene, by X-gal staining. ( A ) overview of the face and forelimb musculature at E14.5. Overall, constitutive ablation of Fat1 causes developmental abnormalities of muscle shape, affecting selective subcutaneous muscles in the face (Zyg. Min and Zyg maj, muscles, Occip. F, orbic. Or. and temporalis Muscles) and selective muscles in the scapulohumeral region. Muscle names are indicated. Muscles which are reduced or show an altered shape have their name underlined in Fat1 ΔTM/ΔTM mutant pictures. Ectopic muscles are indicated with red arrows. ( B ) Muscles of the scapulohumeral area at E14.5 and E15.5, visualized with dorsal views of the scapular muscles at E14.5, and side views of the forelimb at E15.5. Dorsal views reveal the reduced extent of the CM and Rhomboid muscles, and the abnormal connections between the upper and lower parts of the trapezius (amT and spT, respectively). A large additional ectopic muscle (red dotted line, bottom picture) is observed in Fat1 ΔTM/ΔTM embryo, that appears ectopically inserted between the spinodeltoid and Triceps brachii (LoTB and LaTb) muscles. ( C ) Analysis of muscles in the face at E14.5 ( A ), E15.5 ( C , top), and at P0 ( C , bottom), reveals abnormalities in shape, myofibre orientation and density in several subcutaneous muscles (red arrows) that occupy positions equivalent to that of human muscles of facial expression, while deeper muscles such as the masseters (see Figure 6D and data not shown) display normal shape. Overall the topography of muscles affected in Fat1 mutant mice resembles the map of muscles affected in human FSHD muscle in early phases of the disease. Muscle names abbreviations: amT: acromiotrapezius; amd: acromiodeltoid; Bra: brachialis; CM: cutaneous maximus; Ecu: Extensor carpi ulnaris; Ecr: Extensor carpi radialis; Edc: Extensor digitorum communis; Edl: extensor digitorum longus; Fr: Frontalis; LaTb: lateral Triceps Brachii; LoTb: Longitudinal Triceps Brachii; Occ: occipitalis; Orbic. Oc: orbicularis oculis; Orbic Or: Orbicularis Oris; Risor: Risorius (position equivalent to that of Risorius in human); SpD: spinodeltoid; SpT: spinotrapezius; SpTS: Subcutaneous part of the Spinotrapezius muscle; Temp: Temporo-parietal muscle; Zyg: Zygomaticus (position inferred from equivalent position in human).

    Journal: PLoS Genetics

    Article Title: Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy

    doi: 10.1371/journal.pgen.1003550

    Figure Lengend Snippet: Fat1 loss of function alters shapes of selective facial and scapulohumeral muscles. Skeletal muscle groups were visualized in E14.5, E15.5, and E18.5 wild type and Fat1 ΔTM/ΔTM embryos carrying the MLC3f-2E (LacZ) transgene, by X-gal staining. ( A ) overview of the face and forelimb musculature at E14.5. Overall, constitutive ablation of Fat1 causes developmental abnormalities of muscle shape, affecting selective subcutaneous muscles in the face (Zyg. Min and Zyg maj, muscles, Occip. F, orbic. Or. and temporalis Muscles) and selective muscles in the scapulohumeral region. Muscle names are indicated. Muscles which are reduced or show an altered shape have their name underlined in Fat1 ΔTM/ΔTM mutant pictures. Ectopic muscles are indicated with red arrows. ( B ) Muscles of the scapulohumeral area at E14.5 and E15.5, visualized with dorsal views of the scapular muscles at E14.5, and side views of the forelimb at E15.5. Dorsal views reveal the reduced extent of the CM and Rhomboid muscles, and the abnormal connections between the upper and lower parts of the trapezius (amT and spT, respectively). A large additional ectopic muscle (red dotted line, bottom picture) is observed in Fat1 ΔTM/ΔTM embryo, that appears ectopically inserted between the spinodeltoid and Triceps brachii (LoTB and LaTb) muscles. ( C ) Analysis of muscles in the face at E14.5 ( A ), E15.5 ( C , top), and at P0 ( C , bottom), reveals abnormalities in shape, myofibre orientation and density in several subcutaneous muscles (red arrows) that occupy positions equivalent to that of human muscles of facial expression, while deeper muscles such as the masseters (see Figure 6D and data not shown) display normal shape. Overall the topography of muscles affected in Fat1 mutant mice resembles the map of muscles affected in human FSHD muscle in early phases of the disease. Muscle names abbreviations: amT: acromiotrapezius; amd: acromiodeltoid; Bra: brachialis; CM: cutaneous maximus; Ecu: Extensor carpi ulnaris; Ecr: Extensor carpi radialis; Edc: Extensor digitorum communis; Edl: extensor digitorum longus; Fr: Frontalis; LaTb: lateral Triceps Brachii; LoTb: Longitudinal Triceps Brachii; Occ: occipitalis; Orbic. Oc: orbicularis oculis; Orbic Or: Orbicularis Oris; Risor: Risorius (position equivalent to that of Risorius in human); SpD: spinodeltoid; SpT: spinotrapezius; SpTS: Subcutaneous part of the Spinotrapezius muscle; Temp: Temporo-parietal muscle; Zyg: Zygomaticus (position inferred from equivalent position in human).

    Article Snippet: Expression of the human FAT1 gene was monitored by a real time quantitative RT-PCR method using TaqMan gene expression assay reference number Hs00170627_m1 targeting the 5′ part of the FAT1 sequence (Applied biosystem), or using real-time sybrgreen PCR assay (Roche) (see primers below).

    Techniques: Staining, Mutagenesis, Single-particle Tracking, Expressing, Mouse Assay

    Treatment of membranes with phalloidin increases the sedimentability of p205, as well as actin. ( A ) Neutrophil plasma membranes treated without (−) or with (+) phalloidin were solubilized in TEB and fractionated on 20–55% sucrose gradients. The initial membrane extract ( Load ) and gradient fractions (lanes 1–17 ) were analyzed for the presence of cytoskeletal proteins as described in Materials and Methods. Positions of calibration standards ( 9S , 19S , and 30S ) are indicated. ( B ) Phalloidin- induced shift in the sedimentability of p205. Average distribution of p205 in gradient fractions, expressed as a percent of the total F-actin binding at 205 kD on F-actin blot overlays ( n = 3). Similar results were observed when blot strips were re-probed with anti-pepA antibody, indicating that a single 205-kD F-actin–binding polypeptide is present in the 13S, 26S, and 30S complexes.

    Journal: The Journal of Cell Biology

    Article Title: Supervillin (p205): A Novel Membrane-associated, F-Actin-binding Protein in the Villin/Gelsolin Superfamily

    doi:

    Figure Lengend Snippet: Treatment of membranes with phalloidin increases the sedimentability of p205, as well as actin. ( A ) Neutrophil plasma membranes treated without (−) or with (+) phalloidin were solubilized in TEB and fractionated on 20–55% sucrose gradients. The initial membrane extract ( Load ) and gradient fractions (lanes 1–17 ) were analyzed for the presence of cytoskeletal proteins as described in Materials and Methods. Positions of calibration standards ( 9S , 19S , and 30S ) are indicated. ( B ) Phalloidin- induced shift in the sedimentability of p205. Average distribution of p205 in gradient fractions, expressed as a percent of the total F-actin binding at 205 kD on F-actin blot overlays ( n = 3). Similar results were observed when blot strips were re-probed with anti-pepA antibody, indicating that a single 205-kD F-actin–binding polypeptide is present in the 13S, 26S, and 30S complexes.

    Article Snippet: Polyclonal antisera were generated against synthetic peptides corresponding to the two longest p205 sequences (SPVELDEDFDVIFDPYAPR and VPRPQTTAGDVLDGVN) by Research Genetics, Inc. (Huntsville, AL).

    Techniques: Binding Assay

    Distribution of p205 ( A and C ) and E-cadherin ( B and D ) in MDBK cells grown at low cell density. Cells were double labeled with affinity-purified, rabbit anti-pepA IgG and with monoclonal anticadherin antibodies, as described in Materials and Methods. Regions of p205 and cadherin colocalization at the plasma membrane ( white arrows ), and regions of anti-pepA staining alone ( hollow arrows ) are indicated. Bar, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Supervillin (p205): A Novel Membrane-associated, F-Actin-binding Protein in the Villin/Gelsolin Superfamily

    doi:

    Figure Lengend Snippet: Distribution of p205 ( A and C ) and E-cadherin ( B and D ) in MDBK cells grown at low cell density. Cells were double labeled with affinity-purified, rabbit anti-pepA IgG and with monoclonal anticadherin antibodies, as described in Materials and Methods. Regions of p205 and cadherin colocalization at the plasma membrane ( white arrows ), and regions of anti-pepA staining alone ( hollow arrows ) are indicated. Bar, 10 μm.

    Article Snippet: Polyclonal antisera were generated against synthetic peptides corresponding to the two longest p205 sequences (SPVELDEDFDVIFDPYAPR and VPRPQTTAGDVLDGVN) by Research Genetics, Inc. (Huntsville, AL).

    Techniques: Labeling, Affinity Purification, Staining

    p205 is present in many cell types but is not the only ∼205-kD F-actin–binding protein. ( A ) Nitrocellulose blots of HeLa cells (lane 1 ), MDBK cells (lane 2 ), and bovine neutrophils (lane 3 ) stained with affinity-purified anti-pepA antibodies. Only the neutrophils contain a cross-reactive protein at ∼90 kD ( arrowhead ). This 90-kD protein cofractionates with pooled neutrophil granules ( 66 ), specialized vesicles involved in host defense, and is distinct from the ∼90-kD cytosolic actin-binding protein in Fig. 1 C , lane 3. ( B ) Blots of HeLa (lane 1 ), SHSY5Y neuroblastoma (lane 2 ), 3T3 (lane 3 ), MDBK (lane 4 ), NRK (lane 5 ), LLC-PK1 (lane 6 ), and COS-7 cells (lane 7 ) were stained in parallel with antibodies to pepA and F-actin. A higher molecular mass protein that binds F-actin, but not anti-pepA, is observed in lanes 3 and 5 (*).

    Journal: The Journal of Cell Biology

    Article Title: Supervillin (p205): A Novel Membrane-associated, F-Actin-binding Protein in the Villin/Gelsolin Superfamily

    doi:

    Figure Lengend Snippet: p205 is present in many cell types but is not the only ∼205-kD F-actin–binding protein. ( A ) Nitrocellulose blots of HeLa cells (lane 1 ), MDBK cells (lane 2 ), and bovine neutrophils (lane 3 ) stained with affinity-purified anti-pepA antibodies. Only the neutrophils contain a cross-reactive protein at ∼90 kD ( arrowhead ). This 90-kD protein cofractionates with pooled neutrophil granules ( 66 ), specialized vesicles involved in host defense, and is distinct from the ∼90-kD cytosolic actin-binding protein in Fig. 1 C , lane 3. ( B ) Blots of HeLa (lane 1 ), SHSY5Y neuroblastoma (lane 2 ), 3T3 (lane 3 ), MDBK (lane 4 ), NRK (lane 5 ), LLC-PK1 (lane 6 ), and COS-7 cells (lane 7 ) were stained in parallel with antibodies to pepA and F-actin. A higher molecular mass protein that binds F-actin, but not anti-pepA, is observed in lanes 3 and 5 (*).

    Article Snippet: Polyclonal antisera were generated against synthetic peptides corresponding to the two longest p205 sequences (SPVELDEDFDVIFDPYAPR and VPRPQTTAGDVLDGVN) by Research Genetics, Inc. (Huntsville, AL).

    Techniques: Binding Assay, Staining, Affinity Purification

    Distribution of p205 ( A and C) and E-cadherin (B and D ) in MDBK cells grown to high cell density. Cells were double labeled with affinity-purified, rabbit anti-pepA IgG and with monoclonal anticadherin antibodies. Regions of p205 and cadherin colocalization at the plasma membrane ( white arrows ) and regions of anti-pepA staining alone ( hollow arrows ) are indicated. Bar, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Supervillin (p205): A Novel Membrane-associated, F-Actin-binding Protein in the Villin/Gelsolin Superfamily

    doi:

    Figure Lengend Snippet: Distribution of p205 ( A and C) and E-cadherin (B and D ) in MDBK cells grown to high cell density. Cells were double labeled with affinity-purified, rabbit anti-pepA IgG and with monoclonal anticadherin antibodies. Regions of p205 and cadherin colocalization at the plasma membrane ( white arrows ) and regions of anti-pepA staining alone ( hollow arrows ) are indicated. Bar, 10 μm.

    Article Snippet: Polyclonal antisera were generated against synthetic peptides corresponding to the two longest p205 sequences (SPVELDEDFDVIFDPYAPR and VPRPQTTAGDVLDGVN) by Research Genetics, Inc. (Huntsville, AL).

    Techniques: Labeling, Affinity Purification, Staining

    Predicted sequence and domain structure of bovine p205 (supervillin). ( A ) The amino acid sequence starting with the first methionine of the translated ORF includes all eight peptides obtained from purified p205 ( double underline ). The NH 2 -terminal half contains four putative nuclear targeting signals ( gray boxes ) the longest of which resembles the nucleoplasmin targeting signal ( single underline ). The COOH-terminal half of the molecule contains a potential tyrosine phosphorylation site ( black ). Amino acid positions are indicated by numbers in the left margin. These sequence data are available from GenBank/EMBL/DDBJ under accession number AF025996 . ( B ) Schematic representation of the domain structure showing the NH 2 -terminal region with putative nuclear targeting regions ( gray boxes ) juxtaposed with potential protein kinase A phosphorylation sites ( asterisks ). The COOH-terminal domain ( cross-hatched ) shows extensive similarity to villin and gelsolin, with three regions of especially high homology that correspond to potential F-actin–binding sites ( black boxes ). The potential tyrosine phosphorylation site is indicated (·).

    Journal: The Journal of Cell Biology

    Article Title: Supervillin (p205): A Novel Membrane-associated, F-Actin-binding Protein in the Villin/Gelsolin Superfamily

    doi:

    Figure Lengend Snippet: Predicted sequence and domain structure of bovine p205 (supervillin). ( A ) The amino acid sequence starting with the first methionine of the translated ORF includes all eight peptides obtained from purified p205 ( double underline ). The NH 2 -terminal half contains four putative nuclear targeting signals ( gray boxes ) the longest of which resembles the nucleoplasmin targeting signal ( single underline ). The COOH-terminal half of the molecule contains a potential tyrosine phosphorylation site ( black ). Amino acid positions are indicated by numbers in the left margin. These sequence data are available from GenBank/EMBL/DDBJ under accession number AF025996 . ( B ) Schematic representation of the domain structure showing the NH 2 -terminal region with putative nuclear targeting regions ( gray boxes ) juxtaposed with potential protein kinase A phosphorylation sites ( asterisks ). The COOH-terminal domain ( cross-hatched ) shows extensive similarity to villin and gelsolin, with three regions of especially high homology that correspond to potential F-actin–binding sites ( black boxes ). The potential tyrosine phosphorylation site is indicated (·).

    Article Snippet: Polyclonal antisera were generated against synthetic peptides corresponding to the two longest p205 sequences (SPVELDEDFDVIFDPYAPR and VPRPQTTAGDVLDGVN) by Research Genetics, Inc. (Huntsville, AL).

    Techniques: Sequencing, Purification, Binding Assay

    p205 is a peripheral component of the neutrophil plasma membrane skeleton. ( A ) Neutrophil plasma membranes were extracted with either buffer alone (lane 1 ), or buffer containing a final concentration of 2.5 mM MgATP (lane 2 ), 0.25 M KCl (lane 3 ), 1.0 M KCl (lane 4 ), 0.1 M sodium carbonate (lane 5 ), or 0.1 M NaOH (lane 6 ). For each extraction condition, high speed supernatants ( S ) and pellets ( P ) from 130-μg membranes were electrophoresed, blotted, and then probed with 125 I-labeled F-actin and with specific antibodies. ( B ) Neutrophil plasma membranes (100 μg per treatment) were extracted with either buffer alone (lane 1 ), or buffer containing 1% Triton X-100 (lane 2 ), 1% Triton X-100, 50 mM NaCl (lane 3 ), 1% Triton X-100, 250 mM NaCl (lane 4 ), 3% octylglucoside, 250 mM NaCl (lane 5 ), or 0.1% SDS (lane 6 ) and processed as above. A positive control consisted of membranes extracted with 1% SDS at 70°C for 10 min (lane 7 ). The higher mobility F-actin–binding polypeptide present in B is consistently observed after detergent treatment; this band also reacts with an antibody against p205 sequences (see below), suggesting a close structural relationship with p205.

    Journal: The Journal of Cell Biology

    Article Title: Supervillin (p205): A Novel Membrane-associated, F-Actin-binding Protein in the Villin/Gelsolin Superfamily

    doi:

    Figure Lengend Snippet: p205 is a peripheral component of the neutrophil plasma membrane skeleton. ( A ) Neutrophil plasma membranes were extracted with either buffer alone (lane 1 ), or buffer containing a final concentration of 2.5 mM MgATP (lane 2 ), 0.25 M KCl (lane 3 ), 1.0 M KCl (lane 4 ), 0.1 M sodium carbonate (lane 5 ), or 0.1 M NaOH (lane 6 ). For each extraction condition, high speed supernatants ( S ) and pellets ( P ) from 130-μg membranes were electrophoresed, blotted, and then probed with 125 I-labeled F-actin and with specific antibodies. ( B ) Neutrophil plasma membranes (100 μg per treatment) were extracted with either buffer alone (lane 1 ), or buffer containing 1% Triton X-100 (lane 2 ), 1% Triton X-100, 50 mM NaCl (lane 3 ), 1% Triton X-100, 250 mM NaCl (lane 4 ), 3% octylglucoside, 250 mM NaCl (lane 5 ), or 0.1% SDS (lane 6 ) and processed as above. A positive control consisted of membranes extracted with 1% SDS at 70°C for 10 min (lane 7 ). The higher mobility F-actin–binding polypeptide present in B is consistently observed after detergent treatment; this band also reacts with an antibody against p205 sequences (see below), suggesting a close structural relationship with p205.

    Article Snippet: Polyclonal antisera were generated against synthetic peptides corresponding to the two longest p205 sequences (SPVELDEDFDVIFDPYAPR and VPRPQTTAGDVLDGVN) by Research Genetics, Inc. (Huntsville, AL).

    Techniques: Concentration Assay, Labeling, Positive Control, Binding Assay

    Purification of p205 by SDS-PAGE. ( A ) Neutrophil plasma membranes (lane 1 ) and Triton X-100–insoluble pellets (lanes 2–5 ) were separated on a 5% polyacrylamide gel and stained with silver (lanes 1 and 2 ), or electrotransfered to nitrocellulose and probed with either 125 I-labeled F-actin (lane 3 ), or with antibodies against myosin II (lane 4 ), or nonerythroid spectrin/fodrin (lane 5 ). Loads represent 100 μg membranes or equivalent amounts of Triton X-100–insoluble pellets. The location of p205 is indicated ( arrowheads ). ( B ) Eight microsequences were obtained from proteolytic digests of SDS-PAGE–purified p205. Polyclonal rabbit antibodies were generated against synthetic peptides corresponding to two of these sequences ( pepA and pepB ). Residues at variance with the deduced amino acid sequence (Fig. 11 A ) are underlined; a lysine deduced from the cleavage specificity of Endo-LysC is shown in parentheses.

    Journal: The Journal of Cell Biology

    Article Title: Supervillin (p205): A Novel Membrane-associated, F-Actin-binding Protein in the Villin/Gelsolin Superfamily

    doi:

    Figure Lengend Snippet: Purification of p205 by SDS-PAGE. ( A ) Neutrophil plasma membranes (lane 1 ) and Triton X-100–insoluble pellets (lanes 2–5 ) were separated on a 5% polyacrylamide gel and stained with silver (lanes 1 and 2 ), or electrotransfered to nitrocellulose and probed with either 125 I-labeled F-actin (lane 3 ), or with antibodies against myosin II (lane 4 ), or nonerythroid spectrin/fodrin (lane 5 ). Loads represent 100 μg membranes or equivalent amounts of Triton X-100–insoluble pellets. The location of p205 is indicated ( arrowheads ). ( B ) Eight microsequences were obtained from proteolytic digests of SDS-PAGE–purified p205. Polyclonal rabbit antibodies were generated against synthetic peptides corresponding to two of these sequences ( pepA and pepB ). Residues at variance with the deduced amino acid sequence (Fig. 11 A ) are underlined; a lysine deduced from the cleavage specificity of Endo-LysC is shown in parentheses.

    Article Snippet: Polyclonal antisera were generated against synthetic peptides corresponding to the two longest p205 sequences (SPVELDEDFDVIFDPYAPR and VPRPQTTAGDVLDGVN) by Research Genetics, Inc. (Huntsville, AL).

    Techniques: Purification, SDS Page, Staining, Labeling, Generated, Sequencing

    F-actin blot overlay shows that p205 is specifically immunoprecipitated from bovine neutrophil plasma membranes by antibodies against p205 peptides, pepA, and pepB. Proteins were immunoprecipitated from SDS-solubilized, Triton X-100–insoluble pellets by preimmune ( Preimmune ) or immune ( Immune ) sera from four different rabbits (lanes 1–4 ) that were immunized with either peptide A (lanes 1 and 2 ) or peptide B (lanes 3 and 4 ). Antibody specificity is indicated by the absence of p205 from immunoprecipitates generated either with preimmune sera or with immune sera plus the appropriate competing peptide ( Immune + pepA / pepB ). S , p205 in the initial RIPA supernatant.

    Journal: The Journal of Cell Biology

    Article Title: Supervillin (p205): A Novel Membrane-associated, F-Actin-binding Protein in the Villin/Gelsolin Superfamily

    doi:

    Figure Lengend Snippet: F-actin blot overlay shows that p205 is specifically immunoprecipitated from bovine neutrophil plasma membranes by antibodies against p205 peptides, pepA, and pepB. Proteins were immunoprecipitated from SDS-solubilized, Triton X-100–insoluble pellets by preimmune ( Preimmune ) or immune ( Immune ) sera from four different rabbits (lanes 1–4 ) that were immunized with either peptide A (lanes 1 and 2 ) or peptide B (lanes 3 and 4 ). Antibody specificity is indicated by the absence of p205 from immunoprecipitates generated either with preimmune sera or with immune sera plus the appropriate competing peptide ( Immune + pepA / pepB ). S , p205 in the initial RIPA supernatant.

    Article Snippet: Polyclonal antisera were generated against synthetic peptides corresponding to the two longest p205 sequences (SPVELDEDFDVIFDPYAPR and VPRPQTTAGDVLDGVN) by Research Genetics, Inc. (Huntsville, AL).

    Techniques: Immunoprecipitation, Generated

    p205 cosediments with endogenous, phalloidin-stabilized actin ( A ), and actin coimmunoprecipitates with p205 ( B ). ( A ) Neutrophil plasma membranes (lane 1 ) were treated with either fluorescein-phalloidin (lanes 2 and 3 ) or unlabeled phalloidin (lane 4 ), solubilized in TEB, and then incubated with nonspecific IgG (lane 2 ) or antifluorescein IgG (lanes 3 and 4 ) bound to protein A–agarose. p205 was visualized by staining with affinity-purified pepA IgG and by F-actin blot overlays (not shown). ( B ) IgG and actin in immunoprecipitates generated with either nonspecific rabbit IgG (lane 1 ) or affinity-purified, anti-pepA antibody (lane 2 ) after three washes with RIPA buffer. The relative amounts of actin cited in the text were normalized by reference to the amounts of IgG visualized by labeling with radiolabeled secondary IgG.

    Journal: The Journal of Cell Biology

    Article Title: Supervillin (p205): A Novel Membrane-associated, F-Actin-binding Protein in the Villin/Gelsolin Superfamily

    doi:

    Figure Lengend Snippet: p205 cosediments with endogenous, phalloidin-stabilized actin ( A ), and actin coimmunoprecipitates with p205 ( B ). ( A ) Neutrophil plasma membranes (lane 1 ) were treated with either fluorescein-phalloidin (lanes 2 and 3 ) or unlabeled phalloidin (lane 4 ), solubilized in TEB, and then incubated with nonspecific IgG (lane 2 ) or antifluorescein IgG (lanes 3 and 4 ) bound to protein A–agarose. p205 was visualized by staining with affinity-purified pepA IgG and by F-actin blot overlays (not shown). ( B ) IgG and actin in immunoprecipitates generated with either nonspecific rabbit IgG (lane 1 ) or affinity-purified, anti-pepA antibody (lane 2 ) after three washes with RIPA buffer. The relative amounts of actin cited in the text were normalized by reference to the amounts of IgG visualized by labeling with radiolabeled secondary IgG.

    Article Snippet: Polyclonal antisera were generated against synthetic peptides corresponding to the two longest p205 sequences (SPVELDEDFDVIFDPYAPR and VPRPQTTAGDVLDGVN) by Research Genetics, Inc. (Huntsville, AL).

    Techniques: Incubation, Staining, Affinity Purification, Generated, Labeling

    Neutrophil plasma membranes are highly enriched in p205. Fractions highly enriched in plasma membranes ( A ) or secretory vesicles ( B ) were analyzed for the presence of p205 by F-actin blot overlay ( C ). Aliquots (100 μg protein) were fractionated on a 5% SDS–polyacrylamide gel, transferred to nitrocellulose, and then probed with 125 I-labeled F-actin. Lane 1 , plasma membranes; lane 2 , secretory vesicles; and lane 3 , cytosol. E/M , the position of ezrin and moesin. Other cytosolic actin-binding proteins also are detected by this method (lane 3 ).

    Journal: The Journal of Cell Biology

    Article Title: Supervillin (p205): A Novel Membrane-associated, F-Actin-binding Protein in the Villin/Gelsolin Superfamily

    doi:

    Figure Lengend Snippet: Neutrophil plasma membranes are highly enriched in p205. Fractions highly enriched in plasma membranes ( A ) or secretory vesicles ( B ) were analyzed for the presence of p205 by F-actin blot overlay ( C ). Aliquots (100 μg protein) were fractionated on a 5% SDS–polyacrylamide gel, transferred to nitrocellulose, and then probed with 125 I-labeled F-actin. Lane 1 , plasma membranes; lane 2 , secretory vesicles; and lane 3 , cytosol. E/M , the position of ezrin and moesin. Other cytosolic actin-binding proteins also are detected by this method (lane 3 ).

    Article Snippet: Polyclonal antisera were generated against synthetic peptides corresponding to the two longest p205 sequences (SPVELDEDFDVIFDPYAPR and VPRPQTTAGDVLDGVN) by Research Genetics, Inc. (Huntsville, AL).

    Techniques: Labeling, Binding Assay

    Colocalization of p205 ( A , C , and E ) with F-actin ( B ) and cadherin ( D and F ) in ringlike structures and cytoplasmic aggregates 10 min after EGTA treatment to disrupt cell adhesions. After 30 min ( E and F ), most of the cadherin is dissociated from p205, which is diffusely distributed in cytoplasmic puncta. Cells were double labeled with affinity-purified pepA antibodies and either fluorescein-phalloidin or anticadherin antibodies. Bar, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Supervillin (p205): A Novel Membrane-associated, F-Actin-binding Protein in the Villin/Gelsolin Superfamily

    doi:

    Figure Lengend Snippet: Colocalization of p205 ( A , C , and E ) with F-actin ( B ) and cadherin ( D and F ) in ringlike structures and cytoplasmic aggregates 10 min after EGTA treatment to disrupt cell adhesions. After 30 min ( E and F ), most of the cadherin is dissociated from p205, which is diffusely distributed in cytoplasmic puncta. Cells were double labeled with affinity-purified pepA antibodies and either fluorescein-phalloidin or anticadherin antibodies. Bar, 10 μm.

    Article Snippet: Polyclonal antisera were generated against synthetic peptides corresponding to the two longest p205 sequences (SPVELDEDFDVIFDPYAPR and VPRPQTTAGDVLDGVN) by Research Genetics, Inc. (Huntsville, AL).

    Techniques: Labeling, Affinity Purification

    The number of OE progenitors in the cell cycle or S-phase, as well as cell cycle length, is unaltered in Insm1 -/- embryos at E12.5 . (A) At E12.5 there is no difference in the number of OE cells in the cell cycle (those expressing Ki67; P = 0.91) or in S-phase (those having incorporated bromodeoxyuridine (BrdU) 30 minutes after injection; P = 0.29) between Insm1 -/- (grey bars) and Insm1 +/+ littermates (white bars; n = 3 embryo pairs, total of 9 aligned section pairs). (B) The fraction of dividing cells (Ki67+) that are in S-phase (BrdU+), which is indicative of cell cycle length, does not differ between Insm1 -/- mice and their Insm1 +/+ littermates ( P = 0.11). Cell cycle length does not differ among genotypes when considering all progenitors (apical + basal; P = 0.11) or only the basal ones ( P = 0.19). (C) For BrdU incorporation, a larger data set (n = 6 embryo pairs, total of 18 aligned section pairs) also shows no significant difference of cells in S-phase at E12.5 ( P = 0.08). Data are presented as mean values ± standard error of the mean (SEM).

    Journal: Neural Development

    Article Title: Insm1 promotes the transition of olfactory progenitors from apical and proliferative to basal, terminally dividing and neuronogenic

    doi: 10.1186/1749-8104-6-6

    Figure Lengend Snippet: The number of OE progenitors in the cell cycle or S-phase, as well as cell cycle length, is unaltered in Insm1 -/- embryos at E12.5 . (A) At E12.5 there is no difference in the number of OE cells in the cell cycle (those expressing Ki67; P = 0.91) or in S-phase (those having incorporated bromodeoxyuridine (BrdU) 30 minutes after injection; P = 0.29) between Insm1 -/- (grey bars) and Insm1 +/+ littermates (white bars; n = 3 embryo pairs, total of 9 aligned section pairs). (B) The fraction of dividing cells (Ki67+) that are in S-phase (BrdU+), which is indicative of cell cycle length, does not differ between Insm1 -/- mice and their Insm1 +/+ littermates ( P = 0.11). Cell cycle length does not differ among genotypes when considering all progenitors (apical + basal; P = 0.11) or only the basal ones ( P = 0.19). (C) For BrdU incorporation, a larger data set (n = 6 embryo pairs, total of 18 aligned section pairs) also shows no significant difference of cells in S-phase at E12.5 ( P = 0.08). Data are presented as mean values ± standard error of the mean (SEM).

    Article Snippet: Due to the GC-rich nucleoside content of the Insm1 sequence, 1M betaine (B2629, Sigma-Aldrich, St Louis, MO, USA) was added to the reaction mixture to promote DNA melting.

    Techniques: Expressing, Injection, Mouse Assay, BrdU Incorporation Assay

    Decrease of OE progenitors undergoing terminal division at E12.5 in Insm1 -/- mice . (A) Schematic of strategy for detecting terminally dividing cells within the embryonic OE. Pregnant dams were injected with 10 mM EdU at E12.5, followed by 10 mM BrdU 12 hours later at E13. Embryos were sacrificed at E14.5 and processed for immunohistochemical detection of BrdU and chemical detection of EdU. Cells produced by terminal division at E12.5 would be positive for EdU and negative for BrdU (green only; in the schematic as drawn, two cells out of the six). (B) Insm1 -/- mice had an average of 33% fewer terminally dividing progenitors than their paired Insm1 +/+ and Insm1 +/- littermates (n = 3 embryo pairs, total of 9 aligned section pairs, P

    Journal: Neural Development

    Article Title: Insm1 promotes the transition of olfactory progenitors from apical and proliferative to basal, terminally dividing and neuronogenic

    doi: 10.1186/1749-8104-6-6

    Figure Lengend Snippet: Decrease of OE progenitors undergoing terminal division at E12.5 in Insm1 -/- mice . (A) Schematic of strategy for detecting terminally dividing cells within the embryonic OE. Pregnant dams were injected with 10 mM EdU at E12.5, followed by 10 mM BrdU 12 hours later at E13. Embryos were sacrificed at E14.5 and processed for immunohistochemical detection of BrdU and chemical detection of EdU. Cells produced by terminal division at E12.5 would be positive for EdU and negative for BrdU (green only; in the schematic as drawn, two cells out of the six). (B) Insm1 -/- mice had an average of 33% fewer terminally dividing progenitors than their paired Insm1 +/+ and Insm1 +/- littermates (n = 3 embryo pairs, total of 9 aligned section pairs, P

    Article Snippet: Due to the GC-rich nucleoside content of the Insm1 sequence, 1M betaine (B2629, Sigma-Aldrich, St Louis, MO, USA) was added to the reaction mixture to promote DNA melting.

    Techniques: Mouse Assay, Injection, Immunohistochemistry, Produced

    Increase in the number of progenitors expressing ASCL1 (MASH1) and decrease in those expressing NEUROD1 in Insm1 -/- embryonic OE . (A-I) Coronal sections of E12.5 wild type OE. Solid lines delineate basal lamina and dashed lines apical edges. (A-C) Antibodies to ASCL1 and NEUROD1 label largely different subsets of progenitors. (D-F) ASCL1 is expressed in cells located across the epithelium (see also (A)), as expected for TAPs transitioning through intermediate cell layers. Some ASCL1-expressing cells at apical and intermediate positions express pH3 (arrowheads), confirming they are progenitors. (G-I) NEUROD1-expressing cells are at the basal edge and 79% of them also express the cell-cycle marker Ki67 (arrows; n = 3 sections of OE from 2 embryos), indicating that NEUROD1 is expressed in basal progenitors. (J) Representation of a TAP, expressing ASCL1 and migrating from the apical airways to the basal lamina, where Ascl1 is downregulated, NeuroD1 is upregulated, and NEUROD1+ cells (INPs) divide terminally to produce neurons ('N'; 'M' represents mitotic cells, G0 postmitotic cells and Su sustentacular cells). (K, L) Increase in ASCL1-expressing cells ( P

    Journal: Neural Development

    Article Title: Insm1 promotes the transition of olfactory progenitors from apical and proliferative to basal, terminally dividing and neuronogenic

    doi: 10.1186/1749-8104-6-6

    Figure Lengend Snippet: Increase in the number of progenitors expressing ASCL1 (MASH1) and decrease in those expressing NEUROD1 in Insm1 -/- embryonic OE . (A-I) Coronal sections of E12.5 wild type OE. Solid lines delineate basal lamina and dashed lines apical edges. (A-C) Antibodies to ASCL1 and NEUROD1 label largely different subsets of progenitors. (D-F) ASCL1 is expressed in cells located across the epithelium (see also (A)), as expected for TAPs transitioning through intermediate cell layers. Some ASCL1-expressing cells at apical and intermediate positions express pH3 (arrowheads), confirming they are progenitors. (G-I) NEUROD1-expressing cells are at the basal edge and 79% of them also express the cell-cycle marker Ki67 (arrows; n = 3 sections of OE from 2 embryos), indicating that NEUROD1 is expressed in basal progenitors. (J) Representation of a TAP, expressing ASCL1 and migrating from the apical airways to the basal lamina, where Ascl1 is downregulated, NeuroD1 is upregulated, and NEUROD1+ cells (INPs) divide terminally to produce neurons ('N'; 'M' represents mitotic cells, G0 postmitotic cells and Su sustentacular cells). (K, L) Increase in ASCL1-expressing cells ( P

    Article Snippet: Due to the GC-rich nucleoside content of the Insm1 sequence, 1M betaine (B2629, Sigma-Aldrich, St Louis, MO, USA) was added to the reaction mixture to promote DNA melting.

    Techniques: Expressing, Marker

    Basal and intermediate progenitors of OE express Insm 1 mRNA . (A) In situ hybridization (ISH) on a coronal section of an E17.5 mouse embryo head reveals Insm1 in basal and intermediate, but not apical, cells of the OE. Subventricular cells of the olfactory bulb (OB) also express Insm1. (B, C) ISH for Insm1 (B) combined with immunohistochemistry for the cell-division marker Ki67 (C) on the same coronal sections of E14.5 OE demonstrates Insm1 expression in few apical (empty arrows) and most intermediate and basal cells. While most apical progenitors (Ki67-positive) do not express Insm1 (arrowheads), intermediate and basal cells in division do express Insm1 (filled arrows). (D, E) ISH for Insm1 (D) combined with immunohistochemistry for the mitotic marker pH3 (E) on the same coronal sections of E12.5 OE demonstrates Insm1 expression in few apical (empty arrows) and most intermediate and basal cells. While most apical progenitors in mitosis do not express Insm1 (arrowheads), intermediate and basal progenitors in mitosis do express Insm1 (filled arrows).

    Journal: Neural Development

    Article Title: Insm1 promotes the transition of olfactory progenitors from apical and proliferative to basal, terminally dividing and neuronogenic

    doi: 10.1186/1749-8104-6-6

    Figure Lengend Snippet: Basal and intermediate progenitors of OE express Insm 1 mRNA . (A) In situ hybridization (ISH) on a coronal section of an E17.5 mouse embryo head reveals Insm1 in basal and intermediate, but not apical, cells of the OE. Subventricular cells of the olfactory bulb (OB) also express Insm1. (B, C) ISH for Insm1 (B) combined with immunohistochemistry for the cell-division marker Ki67 (C) on the same coronal sections of E14.5 OE demonstrates Insm1 expression in few apical (empty arrows) and most intermediate and basal cells. While most apical progenitors (Ki67-positive) do not express Insm1 (arrowheads), intermediate and basal cells in division do express Insm1 (filled arrows). (D, E) ISH for Insm1 (D) combined with immunohistochemistry for the mitotic marker pH3 (E) on the same coronal sections of E12.5 OE demonstrates Insm1 expression in few apical (empty arrows) and most intermediate and basal cells. While most apical progenitors in mitosis do not express Insm1 (arrowheads), intermediate and basal progenitors in mitosis do express Insm1 (filled arrows).

    Article Snippet: Due to the GC-rich nucleoside content of the Insm1 sequence, 1M betaine (B2629, Sigma-Aldrich, St Louis, MO, USA) was added to the reaction mixture to promote DNA melting.

    Techniques: In Situ Hybridization, Immunohistochemistry, Marker, Expressing

    Increase in the number of apical cells and concomitant decrease in the number of neuro-basal cells in the OE of Insm1 -/- embryos . Representative DAPI stained images from coronal sections of (A, D, H) Insm1 +/+ embryos and (B, E, I) anatomically aligned sections from paired Insm1 -/- littermates. Solid lines indicate the apical (top) and basal (bottom) margins of the OE, and dashed lines indicate the interior border of the apical compartment. In Insm1 -/- mice at ( D-G ) E14.5 and ( H-K ) E18.5, the number of apical cells per (100 μm) 2 of total OE surface is 57% greater (n = 4 embryo pairs, total of 12 aligned section pairs, P

    Journal: Neural Development

    Article Title: Insm1 promotes the transition of olfactory progenitors from apical and proliferative to basal, terminally dividing and neuronogenic

    doi: 10.1186/1749-8104-6-6

    Figure Lengend Snippet: Increase in the number of apical cells and concomitant decrease in the number of neuro-basal cells in the OE of Insm1 -/- embryos . Representative DAPI stained images from coronal sections of (A, D, H) Insm1 +/+ embryos and (B, E, I) anatomically aligned sections from paired Insm1 -/- littermates. Solid lines indicate the apical (top) and basal (bottom) margins of the OE, and dashed lines indicate the interior border of the apical compartment. In Insm1 -/- mice at ( D-G ) E14.5 and ( H-K ) E18.5, the number of apical cells per (100 μm) 2 of total OE surface is 57% greater (n = 4 embryo pairs, total of 12 aligned section pairs, P

    Article Snippet: Due to the GC-rich nucleoside content of the Insm1 sequence, 1M betaine (B2629, Sigma-Aldrich, St Louis, MO, USA) was added to the reaction mixture to promote DNA melting.

    Techniques: Staining, Mouse Assay

    Fewer cells express markers of young (βIII-tubulin) and mature (olfactory marker protein) olfactory receptor neurons in the OE of Insm1 -/- embryos . (A-D) Immunohistochemistry for βIII-tubulin (red) plus (C-D) olfactory marker protein (OMP; green) in representative, anatomically aligned images from the OE of (A, B) E14.5 and (C, D) E18.5 Insm1 -/- and an Insm1 +/+ littermate embryo. Fewer cells express either marker in the knockout (KO). The apical border is toward the center of each panel, and the basal lamina is toward the periphery. (E) Density of young neurons (number of cells expressing βIII-tubulin per (100 μm) 2 ) in OE at various embryonic stages. In Insm1 -/- mice at E11.5, E12.5, E14.5, and E18.5 the number of young neurons (red) is reduced by an average of 43% (n = 3 embryo pairs, total of 9 aligned section pairs, P

    Journal: Neural Development

    Article Title: Insm1 promotes the transition of olfactory progenitors from apical and proliferative to basal, terminally dividing and neuronogenic

    doi: 10.1186/1749-8104-6-6

    Figure Lengend Snippet: Fewer cells express markers of young (βIII-tubulin) and mature (olfactory marker protein) olfactory receptor neurons in the OE of Insm1 -/- embryos . (A-D) Immunohistochemistry for βIII-tubulin (red) plus (C-D) olfactory marker protein (OMP; green) in representative, anatomically aligned images from the OE of (A, B) E14.5 and (C, D) E18.5 Insm1 -/- and an Insm1 +/+ littermate embryo. Fewer cells express either marker in the knockout (KO). The apical border is toward the center of each panel, and the basal lamina is toward the periphery. (E) Density of young neurons (number of cells expressing βIII-tubulin per (100 μm) 2 ) in OE at various embryonic stages. In Insm1 -/- mice at E11.5, E12.5, E14.5, and E18.5 the number of young neurons (red) is reduced by an average of 43% (n = 3 embryo pairs, total of 9 aligned section pairs, P

    Article Snippet: Due to the GC-rich nucleoside content of the Insm1 sequence, 1M betaine (B2629, Sigma-Aldrich, St Louis, MO, USA) was added to the reaction mixture to promote DNA melting.

    Techniques: Marker, Immunohistochemistry, Knock-Out, Gene Knockout, Expressing, Mouse Assay

    Increased apoptosis in the OE of Insm1 -/- embryos does not precede the decrease in nascent neurons . (A-F) Immunohistochemistry for activated cleaved caspase 3 (ACC3), a marker of apoptotic cells, at selected stages. ACC3+ staining is indicated by arrowheads. (G) At E11.5 (and compare (A) and (B)) and E12.5, there is no significant difference in ACC3+ cells (n = 3 embryo pairs, P = 0.63). By E14.5 (compare (C) and (D)), more ACC3+ cells are detected in Insm1 -/- mice than in their paired Insm1 +/+ littermates (n = 3 embryo pairs, P

    Journal: Neural Development

    Article Title: Insm1 promotes the transition of olfactory progenitors from apical and proliferative to basal, terminally dividing and neuronogenic

    doi: 10.1186/1749-8104-6-6

    Figure Lengend Snippet: Increased apoptosis in the OE of Insm1 -/- embryos does not precede the decrease in nascent neurons . (A-F) Immunohistochemistry for activated cleaved caspase 3 (ACC3), a marker of apoptotic cells, at selected stages. ACC3+ staining is indicated by arrowheads. (G) At E11.5 (and compare (A) and (B)) and E12.5, there is no significant difference in ACC3+ cells (n = 3 embryo pairs, P = 0.63). By E14.5 (compare (C) and (D)), more ACC3+ cells are detected in Insm1 -/- mice than in their paired Insm1 +/+ littermates (n = 3 embryo pairs, P

    Article Snippet: Due to the GC-rich nucleoside content of the Insm1 sequence, 1M betaine (B2629, Sigma-Aldrich, St Louis, MO, USA) was added to the reaction mixture to promote DNA melting.

    Techniques: Immunohistochemistry, Marker, Staining, Mouse Assay

    Mitotically active (pH3+) progenitors decrease basally and increase apically in the OE of Insm1 -/- embryos . (A , D , G) Representative images from coronal sections of Insm1 +/+ mice. (B , E , H) Anatomically aligned section pairs from paired Insm1 -/- littermates. Dorsal is toward the right, ventral toward the left. (A-C) At E10.5, there is no statistically significant difference in pH3+ cells overall, or at either the apical or basal border of the OE (n = 4 embryo pairs, total of 12 aligned section pairs, P = 0.36 overall, P = 0.25 apically, P = 0.57 basally). (D-F) At E12.5, Insm1 -/- mice exhibit no difference in overall numbers of pH3+ (green) cells when compared with Insm1 +/+ littermates, but an average 42% increase in pH3+ cells in the apical subpopulation ( P

    Journal: Neural Development

    Article Title: Insm1 promotes the transition of olfactory progenitors from apical and proliferative to basal, terminally dividing and neuronogenic

    doi: 10.1186/1749-8104-6-6

    Figure Lengend Snippet: Mitotically active (pH3+) progenitors decrease basally and increase apically in the OE of Insm1 -/- embryos . (A , D , G) Representative images from coronal sections of Insm1 +/+ mice. (B , E , H) Anatomically aligned section pairs from paired Insm1 -/- littermates. Dorsal is toward the right, ventral toward the left. (A-C) At E10.5, there is no statistically significant difference in pH3+ cells overall, or at either the apical or basal border of the OE (n = 4 embryo pairs, total of 12 aligned section pairs, P = 0.36 overall, P = 0.25 apically, P = 0.57 basally). (D-F) At E12.5, Insm1 -/- mice exhibit no difference in overall numbers of pH3+ (green) cells when compared with Insm1 +/+ littermates, but an average 42% increase in pH3+ cells in the apical subpopulation ( P

    Article Snippet: Due to the GC-rich nucleoside content of the Insm1 sequence, 1M betaine (B2629, Sigma-Aldrich, St Louis, MO, USA) was added to the reaction mixture to promote DNA melting.

    Techniques: Mouse Assay

    Generation of Insm1 knockout mice . (A) Schematic of the recombination strategy used to generate the Insm1 knockout. Using homologous recombination in embryonic stem (ES) cells, we replaced a genomic DNA fragment of 3.4 kb (in blue/cyan) containing the entire single exon of Insm1 plus 927 bp of upstream and 955 bp of downstream sequence with a 'floxed' neomycin cassette (in red/pink). After generating chimeric mice and, from these, heterozygotes, the neomycin cassette was removed by crossing with E2a-Cre transgenic mice. Probes (5' and 3') used for Southern blot genotyping and DNA fragments obtained by PCR genotyping of the wild type, knockout + neomycin and knockout alleles are depicted. (B) Southern blots of Bgl II digests of genomic DNA from recombined ES cells with 5' and 3' probes reveal the expected bands from the knockout (KO) and wild type alleles and confirm that recombination was complete. (C) Genotyping PCR using genomic DNA obtained from a litter of embryos (embryonic day 10.5) resulting from crossing two heterozygous parents. Primers used are described in methods and their products depicted in (A). The litter contained two knockouts (-/-), two wild types (+/+) and four heterozygotes (+/-), as indicated in the figure. (D) In situ hybridization (ISH) with an antisense probe against the 3' UTR of Insm1 on wild type and knockout littermate embryos (E14.5, coronal sections) reveal the presence of Insm1 mRNA in the brain, retina and olfactory epithelium (arrowheads) of wild type but not of knockout embryos, functionally confirming the knockout of Insm1 . Scale bars: 500 μm.

    Journal: Neural Development

    Article Title: Insm1 promotes the transition of olfactory progenitors from apical and proliferative to basal, terminally dividing and neuronogenic

    doi: 10.1186/1749-8104-6-6

    Figure Lengend Snippet: Generation of Insm1 knockout mice . (A) Schematic of the recombination strategy used to generate the Insm1 knockout. Using homologous recombination in embryonic stem (ES) cells, we replaced a genomic DNA fragment of 3.4 kb (in blue/cyan) containing the entire single exon of Insm1 plus 927 bp of upstream and 955 bp of downstream sequence with a 'floxed' neomycin cassette (in red/pink). After generating chimeric mice and, from these, heterozygotes, the neomycin cassette was removed by crossing with E2a-Cre transgenic mice. Probes (5' and 3') used for Southern blot genotyping and DNA fragments obtained by PCR genotyping of the wild type, knockout + neomycin and knockout alleles are depicted. (B) Southern blots of Bgl II digests of genomic DNA from recombined ES cells with 5' and 3' probes reveal the expected bands from the knockout (KO) and wild type alleles and confirm that recombination was complete. (C) Genotyping PCR using genomic DNA obtained from a litter of embryos (embryonic day 10.5) resulting from crossing two heterozygous parents. Primers used are described in methods and their products depicted in (A). The litter contained two knockouts (-/-), two wild types (+/+) and four heterozygotes (+/-), as indicated in the figure. (D) In situ hybridization (ISH) with an antisense probe against the 3' UTR of Insm1 on wild type and knockout littermate embryos (E14.5, coronal sections) reveal the presence of Insm1 mRNA in the brain, retina and olfactory epithelium (arrowheads) of wild type but not of knockout embryos, functionally confirming the knockout of Insm1 . Scale bars: 500 μm.

    Article Snippet: Due to the GC-rich nucleoside content of the Insm1 sequence, 1M betaine (B2629, Sigma-Aldrich, St Louis, MO, USA) was added to the reaction mixture to promote DNA melting.

    Techniques: Knock-Out, Mouse Assay, Homologous Recombination, Sequencing, Transgenic Assay, Southern Blot, Polymerase Chain Reaction, Gene Knockout, In Situ Hybridization

    Phenotypic similarities between egl-46 mutant nematodes and Insm1 knockout mouse OEs suggest a conserved role for both orthologs in promoting terminal, neuronogenic progenitors . (A) In C. elegans , EGL-46 (green) is expressed in late neuronal progenitors destined to terminally divide to produce two neurons (N/N divisions; some of these daughters undergo programmed cell death and are labeled X) and transiently in the nascent neurons. EGL-46 is not expressed in early neural progenitors, which divide to generate more progenitors (P/P or P/N divisions). (B) In egl-46 mutants, cells that would normally terminally divide instead undergo proliferative divisions, thus delaying terminal, neuronogenic divisions. (C) In the embryonic mammalian OE early progenitor cells divide apically (P/P) to produce more progenitors and/or sustentacular cells (which do not display markers of differentiation until later stages). Some of the apically produced cells migrate to the basal lamina, where they divide again to produce neurons (some of these basal progenitors undergo N/N divisions). By E14.5 apical and neuro-basal cells are distinguished based on their nuclear morphology, which is elongate in the apical layer. By E16.5, apical progenitors are believed to produce sustentacular cells, basal progenitors are believed to produce developing neurons, and these populations are believed to be increasingly separate, with diminishing migration from the apical to the basal population. Cells known to express Insm1 are indicated in purple. (D) Insm1 -/- OEs contain more apical and presumed proliferative progenitors and fewer basal, terminally dividing and presumed neuronogenic progenitors, a phenotype similar to that caused by egl-46 mutation in nematodes (compare (B) and (D)). This leads to a thicker apical layer (delineated by a dashed line in (C, D)) and a thinner neuro-basal layer. In contrast to egl-46 mutant nematodes, Insm1 -/- mouse embryonic OEs display fewer neurons, and at a later stage increased apoptosis. M represents cells in mitosis, N neurons, Su sustentacular cells and QL a neuroblast of the nematode lineage.

    Journal: Neural Development

    Article Title: Insm1 promotes the transition of olfactory progenitors from apical and proliferative to basal, terminally dividing and neuronogenic

    doi: 10.1186/1749-8104-6-6

    Figure Lengend Snippet: Phenotypic similarities between egl-46 mutant nematodes and Insm1 knockout mouse OEs suggest a conserved role for both orthologs in promoting terminal, neuronogenic progenitors . (A) In C. elegans , EGL-46 (green) is expressed in late neuronal progenitors destined to terminally divide to produce two neurons (N/N divisions; some of these daughters undergo programmed cell death and are labeled X) and transiently in the nascent neurons. EGL-46 is not expressed in early neural progenitors, which divide to generate more progenitors (P/P or P/N divisions). (B) In egl-46 mutants, cells that would normally terminally divide instead undergo proliferative divisions, thus delaying terminal, neuronogenic divisions. (C) In the embryonic mammalian OE early progenitor cells divide apically (P/P) to produce more progenitors and/or sustentacular cells (which do not display markers of differentiation until later stages). Some of the apically produced cells migrate to the basal lamina, where they divide again to produce neurons (some of these basal progenitors undergo N/N divisions). By E14.5 apical and neuro-basal cells are distinguished based on their nuclear morphology, which is elongate in the apical layer. By E16.5, apical progenitors are believed to produce sustentacular cells, basal progenitors are believed to produce developing neurons, and these populations are believed to be increasingly separate, with diminishing migration from the apical to the basal population. Cells known to express Insm1 are indicated in purple. (D) Insm1 -/- OEs contain more apical and presumed proliferative progenitors and fewer basal, terminally dividing and presumed neuronogenic progenitors, a phenotype similar to that caused by egl-46 mutation in nematodes (compare (B) and (D)). This leads to a thicker apical layer (delineated by a dashed line in (C, D)) and a thinner neuro-basal layer. In contrast to egl-46 mutant nematodes, Insm1 -/- mouse embryonic OEs display fewer neurons, and at a later stage increased apoptosis. M represents cells in mitosis, N neurons, Su sustentacular cells and QL a neuroblast of the nematode lineage.

    Article Snippet: Due to the GC-rich nucleoside content of the Insm1 sequence, 1M betaine (B2629, Sigma-Aldrich, St Louis, MO, USA) was added to the reaction mixture to promote DNA melting.

    Techniques: Mutagenesis, Knock-Out, Labeling, Produced, Migration

    Relative positions of specific gRNAs in editing cascades for both strains. The minicircle-encoded and maxicircle-encoded gRNAs were mapped to the 12 mature edited maxicircle transcript sequences. The edited sequences (CO3, Cyb, Murf2 and A6) are in green, the editing domains are in orange and the minicircle-encoded gRNAs are in red. Maxicircle-encoded gRNAs are shown as blue arrows labeled “MC gRNA” above the sequences. The gRNAs are labeled with the gene name and Roman numerals which refer to the location in the functional editing cascade; the low numbers indicate roles early in the cascade and the higher numbers roles later in the cascade. Redundant gRNAs are indicated with a, b, etc. To easily compare the extent of gRNA coverage in the two strains, the editing domains for each gene in both strains are extracted together with the gRNA location information and shown side by side. Apparently missing overlapping gRNAs are indicated by blue circles.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Comparison of the Mitochondrial Genomes and Steady State Transcriptomes of Two Strains of the Trypanosomatid Parasite, Leishmania tarentolae

    doi: 10.1371/journal.pntd.0003841

    Figure Lengend Snippet: Relative positions of specific gRNAs in editing cascades for both strains. The minicircle-encoded and maxicircle-encoded gRNAs were mapped to the 12 mature edited maxicircle transcript sequences. The edited sequences (CO3, Cyb, Murf2 and A6) are in green, the editing domains are in orange and the minicircle-encoded gRNAs are in red. Maxicircle-encoded gRNAs are shown as blue arrows labeled “MC gRNA” above the sequences. The gRNAs are labeled with the gene name and Roman numerals which refer to the location in the functional editing cascade; the low numbers indicate roles early in the cascade and the higher numbers roles later in the cascade. Redundant gRNAs are indicated with a, b, etc. To easily compare the extent of gRNA coverage in the two strains, the editing domains for each gene in both strains are extracted together with the gRNA location information and shown side by side. Apparently missing overlapping gRNAs are indicated by blue circles.

    Article Snippet: PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions.

    Techniques: Labeling, Functional Assay

    UC steady state gRNA reads divided by minicircle copy number per network. See Fig 14 legend for details.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Comparison of the Mitochondrial Genomes and Steady State Transcriptomes of Two Strains of the Trypanosomatid Parasite, Leishmania tarentolae

    doi: 10.1371/journal.pntd.0003841

    Figure Lengend Snippet: UC steady state gRNA reads divided by minicircle copy number per network. See Fig 14 legend for details.

    Article Snippet: PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions.

    Techniques:

    FPKM quantitation of minicircle steady state transcripts. LEM125 (light red) and UC (blue) minicircle steady state transcripts as percentage of total minicircle steady state transcripts. The minicircle numbers are shown below and the minicircles are grouped according to the gene the editing of which is mediated by the encoded gRNAs.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Comparison of the Mitochondrial Genomes and Steady State Transcriptomes of Two Strains of the Trypanosomatid Parasite, Leishmania tarentolae

    doi: 10.1371/journal.pntd.0003841

    Figure Lengend Snippet: FPKM quantitation of minicircle steady state transcripts. LEM125 (light red) and UC (blue) minicircle steady state transcripts as percentage of total minicircle steady state transcripts. The minicircle numbers are shown below and the minicircles are grouped according to the gene the editing of which is mediated by the encoded gRNAs.

    Article Snippet: PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions.

    Techniques: Quantitation Assay

    Three representative minicircles mapped with LEM large reads. The light red is + strand and the blue is—strand. The extensive mappings of steady state transcripts from both strands over almost the entire minicircle are shown boxed. See Fig 10 legend for details.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Comparison of the Mitochondrial Genomes and Steady State Transcriptomes of Two Strains of the Trypanosomatid Parasite, Leishmania tarentolae

    doi: 10.1371/journal.pntd.0003841

    Figure Lengend Snippet: Three representative minicircles mapped with LEM large reads. The light red is + strand and the blue is—strand. The extensive mappings of steady state transcripts from both strands over almost the entire minicircle are shown boxed. See Fig 10 legend for details.

    Article Snippet: PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions.

    Techniques:

    Length distribution of minicircles. A. Length distribution of 22,680 randomly single cleaved minicircle DNA molecules sequenced on PacBio platform. B. Length distribution of the final dataset of 114 minicircles.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Comparison of the Mitochondrial Genomes and Steady State Transcriptomes of Two Strains of the Trypanosomatid Parasite, Leishmania tarentolae

    doi: 10.1371/journal.pntd.0003841

    Figure Lengend Snippet: Length distribution of minicircles. A. Length distribution of 22,680 randomly single cleaved minicircle DNA molecules sequenced on PacBio platform. B. Length distribution of the final dataset of 114 minicircles.

    Article Snippet: PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions.

    Techniques:

    FPKM quantitation of minicircle steady state transcripts. Percent expression of steady state total minicircle transcripts compared to percent of LEM125 minicircle DNA per network. Minicircle percent data from are Fig 3 . See Fig 14 legend for details.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Comparison of the Mitochondrial Genomes and Steady State Transcriptomes of Two Strains of the Trypanosomatid Parasite, Leishmania tarentolae

    doi: 10.1371/journal.pntd.0003841

    Figure Lengend Snippet: FPKM quantitation of minicircle steady state transcripts. Percent expression of steady state total minicircle transcripts compared to percent of LEM125 minicircle DNA per network. Minicircle percent data from are Fig 3 . See Fig 14 legend for details.

    Article Snippet: PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions.

    Techniques: Quantitation Assay, Expressing

    LEM125 steady state gRNA reads divided by minicircle copy number per network. See Fig 14 legend for details.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Comparison of the Mitochondrial Genomes and Steady State Transcriptomes of Two Strains of the Trypanosomatid Parasite, Leishmania tarentolae

    doi: 10.1371/journal.pntd.0003841

    Figure Lengend Snippet: LEM125 steady state gRNA reads divided by minicircle copy number per network. See Fig 14 legend for details.

    Article Snippet: PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions.

    Techniques:

    Extracted columns of conserved patterns of nucleotide changes. Nine regions were extracted from an alignment of CCS reads that assembled to a specific minicircle sequence class. Conserved patterns of single nucleotide changes in 14 columns in these regions are highlighted. See Fig 4 legend for details.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Comparison of the Mitochondrial Genomes and Steady State Transcriptomes of Two Strains of the Trypanosomatid Parasite, Leishmania tarentolae

    doi: 10.1371/journal.pntd.0003841

    Figure Lengend Snippet: Extracted columns of conserved patterns of nucleotide changes. Nine regions were extracted from an alignment of CCS reads that assembled to a specific minicircle sequence class. Conserved patterns of single nucleotide changes in 14 columns in these regions are highlighted. See Fig 4 legend for details.

    Article Snippet: PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions.

    Techniques: Sequencing

    Mapping of RNA reads from both strains to the 114 minicircles. Reads from the small mitochondrial RNA libraries were mapped against minicircle sequences using Bowtie. The linearized minicircles from both strains are concatenated head to tail with 20 nt spaces inserted for the mapping and indicated under the maps by dark blue tracts with the mc numbers. The locations of the putative encoded gRNA genes within the minicircles are also annotated by small blue boxes below each minicircle tract. VR refers to the “variable region” of the minicircle. The minicircles are organized in groups of cascades according to the specific gene, the editing of which is mediated by the gRNA, and each minicircle is denoted by the “mc number”. The plus strand is light red and the minus strand blue. Lines were inserted to show that the putative gRNA genes line up with the corresponding peaks of gRNA reads. One gRNA peak (indicated by open arrow) was enlarged for better visualization of the flush 5’ ends and the heterogeneous 3’ ends.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Comparison of the Mitochondrial Genomes and Steady State Transcriptomes of Two Strains of the Trypanosomatid Parasite, Leishmania tarentolae

    doi: 10.1371/journal.pntd.0003841

    Figure Lengend Snippet: Mapping of RNA reads from both strains to the 114 minicircles. Reads from the small mitochondrial RNA libraries were mapped against minicircle sequences using Bowtie. The linearized minicircles from both strains are concatenated head to tail with 20 nt spaces inserted for the mapping and indicated under the maps by dark blue tracts with the mc numbers. The locations of the putative encoded gRNA genes within the minicircles are also annotated by small blue boxes below each minicircle tract. VR refers to the “variable region” of the minicircle. The minicircles are organized in groups of cascades according to the specific gene, the editing of which is mediated by the gRNA, and each minicircle is denoted by the “mc number”. The plus strand is light red and the minus strand blue. Lines were inserted to show that the putative gRNA genes line up with the corresponding peaks of gRNA reads. One gRNA peak (indicated by open arrow) was enlarged for better visualization of the flush 5’ ends and the heterogeneous 3’ ends.

    Article Snippet: PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions.

    Techniques:

    Minicircle sequence classes. The average number of minicircle sequence classes per network was calculated from the number of PacBio CCS reads that assembled to specific minicircles (see Table 2 ). These values are plotted for both strains as number of minicircles of a specific sequence class/network (assuming 10,000 minicircles per network) versus the mc number (see Table 2 ).

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Comparison of the Mitochondrial Genomes and Steady State Transcriptomes of Two Strains of the Trypanosomatid Parasite, Leishmania tarentolae

    doi: 10.1371/journal.pntd.0003841

    Figure Lengend Snippet: Minicircle sequence classes. The average number of minicircle sequence classes per network was calculated from the number of PacBio CCS reads that assembled to specific minicircles (see Table 2 ). These values are plotted for both strains as number of minicircles of a specific sequence class/network (assuming 10,000 minicircles per network) versus the mc number (see Table 2 ).

    Article Snippet: PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions.

    Techniques: Sequencing

    Representative linearized minicircle from L. tarentolae showing several characteristic features. The mc8 minicircle is linearized at the 5’ end of the CSB3 sequence. The “bend” region is a sequence-dependent distortion of the double helix. The encoded gRNA is annotated and is located at a characteristic distance from the CSB3. The 5'-3' polarities of the three CSB sequences, the bend and the gRNA are indicated by arrows. VR indicates Variable Region, CR indicates Conserved Region.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Comparison of the Mitochondrial Genomes and Steady State Transcriptomes of Two Strains of the Trypanosomatid Parasite, Leishmania tarentolae

    doi: 10.1371/journal.pntd.0003841

    Figure Lengend Snippet: Representative linearized minicircle from L. tarentolae showing several characteristic features. The mc8 minicircle is linearized at the 5’ end of the CSB3 sequence. The “bend” region is a sequence-dependent distortion of the double helix. The encoded gRNA is annotated and is located at a characteristic distance from the CSB3. The 5'-3' polarities of the three CSB sequences, the bend and the gRNA are indicated by arrows. VR indicates Variable Region, CR indicates Conserved Region.

    Article Snippet: PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions.

    Techniques: Sequencing

    FPKM quantitation of minicircle steady state transcripts. Percent expression of steady state gRNA transcripts compared to percent of UC minicircle DNA per network. Minicircle percent data are from Fig 3 . See Fig 14 legend for details.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Comparison of the Mitochondrial Genomes and Steady State Transcriptomes of Two Strains of the Trypanosomatid Parasite, Leishmania tarentolae

    doi: 10.1371/journal.pntd.0003841

    Figure Lengend Snippet: FPKM quantitation of minicircle steady state transcripts. Percent expression of steady state gRNA transcripts compared to percent of UC minicircle DNA per network. Minicircle percent data are from Fig 3 . See Fig 14 legend for details.

    Article Snippet: PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions.

    Techniques: Quantitation Assay, Expressing

    UC total minicircle RNA reads divided by minicircle copy number per network. See Fig 14 legend for details.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Comparison of the Mitochondrial Genomes and Steady State Transcriptomes of Two Strains of the Trypanosomatid Parasite, Leishmania tarentolae

    doi: 10.1371/journal.pntd.0003841

    Figure Lengend Snippet: UC total minicircle RNA reads divided by minicircle copy number per network. See Fig 14 legend for details.

    Article Snippet: PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions.

    Techniques:

    Conserved patterns of nucleotide changes. Multiple alignment of 58 CCS sequences which assembled to the minicircle, mc54, which encodes gND8-XII. The minicircle consensus sequence from nt 550–664 is at the top. Dots indicate matches and squiggles indicate deletions. Dashes indicate gaps. Columns with conserved single nucleotide mutations (T to C, A to T, A to G, G to C, C to T, A to G) in 6 of the CCS sequences at identical relative positions are boxed.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Comparison of the Mitochondrial Genomes and Steady State Transcriptomes of Two Strains of the Trypanosomatid Parasite, Leishmania tarentolae

    doi: 10.1371/journal.pntd.0003841

    Figure Lengend Snippet: Conserved patterns of nucleotide changes. Multiple alignment of 58 CCS sequences which assembled to the minicircle, mc54, which encodes gND8-XII. The minicircle consensus sequence from nt 550–664 is at the top. Dots indicate matches and squiggles indicate deletions. Dashes indicate gaps. Columns with conserved single nucleotide mutations (T to C, A to T, A to G, G to C, C to T, A to G) in 6 of the CCS sequences at identical relative positions are boxed.

    Article Snippet: PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions.

    Techniques: Sequencing

    FPKM quantitation of minicircle steady state transcripts. LEM125 and UC steady state gRNA transcripts as percentage of total steady state gRNA transcripts. The names of the gRNAs are shown below and the gRNAs are grouped as in Fig 14 .

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Comparison of the Mitochondrial Genomes and Steady State Transcriptomes of Two Strains of the Trypanosomatid Parasite, Leishmania tarentolae

    doi: 10.1371/journal.pntd.0003841

    Figure Lengend Snippet: FPKM quantitation of minicircle steady state transcripts. LEM125 and UC steady state gRNA transcripts as percentage of total steady state gRNA transcripts. The names of the gRNAs are shown below and the gRNAs are grouped as in Fig 14 .

    Article Snippet: PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions.

    Techniques: Quantitation Assay

    Location of conserved single nucleotide changes in representative minicircle. Diagram of linearized mc1 minicircle showing the relative locations of similar conserved patterns of single nucleotide changes (arrows) as in Fig 5 . CR indicates Conserved Region, VR indicates Variable Region.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Comparison of the Mitochondrial Genomes and Steady State Transcriptomes of Two Strains of the Trypanosomatid Parasite, Leishmania tarentolae

    doi: 10.1371/journal.pntd.0003841

    Figure Lengend Snippet: Location of conserved single nucleotide changes in representative minicircle. Diagram of linearized mc1 minicircle showing the relative locations of similar conserved patterns of single nucleotide changes (arrows) as in Fig 5 . CR indicates Conserved Region, VR indicates Variable Region.

    Article Snippet: PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions.

    Techniques:

    Scatter plots of minicircle steady state RNA versus minicircle copy number for LEM and UC strains. Each minicircle shown in blue with fit line in pink. R = 0.62 for LEM, R = 0.50 for UC.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Comparison of the Mitochondrial Genomes and Steady State Transcriptomes of Two Strains of the Trypanosomatid Parasite, Leishmania tarentolae

    doi: 10.1371/journal.pntd.0003841

    Figure Lengend Snippet: Scatter plots of minicircle steady state RNA versus minicircle copy number for LEM and UC strains. Each minicircle shown in blue with fit line in pink. R = 0.62 for LEM, R = 0.50 for UC.

    Article Snippet: PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions.

    Techniques:

    LEM125 total minicircle RNA reads divided by minicircle copy number per network. See Fig 14 legend for details.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Comparison of the Mitochondrial Genomes and Steady State Transcriptomes of Two Strains of the Trypanosomatid Parasite, Leishmania tarentolae

    doi: 10.1371/journal.pntd.0003841

    Figure Lengend Snippet: LEM125 total minicircle RNA reads divided by minicircle copy number per network. See Fig 14 legend for details.

    Article Snippet: PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions.

    Techniques:

    VF1 antagonizes the innate immune response to MNV infection. (A) Time course of ISG54 and CXCL10 mRNA expression in RAW264.7 cells infected at an MOI of 0.1 TCID 50 per cell. mRNA levels were quantified by qPCR using an endogenous control gene (Hypoxanthine-guanine phosphoribosyltransferase, HPRT). Expression of the respective mRNAs was then calculated using the ΔΔCt method to compare infected and mock infected cells. Relative fold change was calculated using mock infected samples taken at comparable time points. Normalization was performed following MNV quantification in each sample. Infections were carried out in triplicate with each sample being subsequently analyzed in duplicate by qPCR. The error bars denote standard deviation from the mean. Statistical analysis was performed using an unpaired two tailed t test. (B) Time course of IFN-Beta mRNA and protein production in RAW264.7 cells infected as in (A). mRNA upregulation was calculated as described for (A). Interferon Beta protein was quantified by murine IFN-B specific ELISA at 24 hpi from supernatant samples taken from infected cells. For the ELISA infections were carried out in sextuplate. The error bars denote standard deviation from the mean. (C) MEF cells transfected with an IFN-Beta promoter driven luciferase reporter as well as expression constructs for RIG1, MDA5, MAVS or TBK1 were co-transfected with plasmid DNA expressing VF1 or blank message. Luciferase production was assayed 24 hours post transfection as described in the Materials and Methods . Transfections were carried out in triplicate. The error bars denote standard deviation from the mean. Statistical analysis was performed using an unpaired two tailed t test.

    Journal: PLoS Pathogens

    Article Title: Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4

    doi: 10.1371/journal.ppat.1002413

    Figure Lengend Snippet: VF1 antagonizes the innate immune response to MNV infection. (A) Time course of ISG54 and CXCL10 mRNA expression in RAW264.7 cells infected at an MOI of 0.1 TCID 50 per cell. mRNA levels were quantified by qPCR using an endogenous control gene (Hypoxanthine-guanine phosphoribosyltransferase, HPRT). Expression of the respective mRNAs was then calculated using the ΔΔCt method to compare infected and mock infected cells. Relative fold change was calculated using mock infected samples taken at comparable time points. Normalization was performed following MNV quantification in each sample. Infections were carried out in triplicate with each sample being subsequently analyzed in duplicate by qPCR. The error bars denote standard deviation from the mean. Statistical analysis was performed using an unpaired two tailed t test. (B) Time course of IFN-Beta mRNA and protein production in RAW264.7 cells infected as in (A). mRNA upregulation was calculated as described for (A). Interferon Beta protein was quantified by murine IFN-B specific ELISA at 24 hpi from supernatant samples taken from infected cells. For the ELISA infections were carried out in sextuplate. The error bars denote standard deviation from the mean. (C) MEF cells transfected with an IFN-Beta promoter driven luciferase reporter as well as expression constructs for RIG1, MDA5, MAVS or TBK1 were co-transfected with plasmid DNA expressing VF1 or blank message. Luciferase production was assayed 24 hours post transfection as described in the Materials and Methods . Transfections were carried out in triplicate. The error bars denote standard deviation from the mean. Statistical analysis was performed using an unpaired two tailed t test.

    Article Snippet: GFP fusions of VF1 were generated by cloning the VF1 encoding sequence into the GFP plasmids pEGFP-N1 and pEGFP-C1 (Clontech).

    Techniques: Infection, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Transfection, Luciferase, Construct, Plasmid Preparation

    Viruses lacking VF1 are altered in their ability to induce apoptosis. (A) Time course of caspase 3/7 activity in RAW264.7 cells treated with Staurosporine or infected with wild-type MNV-1 (WT) or a VF1 knockout virus (M1). Cells were infected at a MOI of 5 TCID 50 per cell and samples harvested at various times post infection. Caspase 3/7 activity was determined using a commercial assay from Promega (Glo 3/7) and the activity represented as relative light units (RLU). Each assay was performed in triplicate with the average and standard error within a single experiment plotted. Statistical analysis was performed using 2 way ANOVA with Bonfferoni post tests (B) Western blot analysis of extracts prepared from cells infected as detailed in (A). Samples were prepared at various times post infection, separated by SDS-PAGE and subsequently immune blotted using antisera to GAPDH, NS7, VP1 or VF1. Non-infected (Mock) cells were included as controls. (C). Caspase 3/7 activity assay in RAW 264.7 cells infected with M1 or WT MNV-1 or UV-inactivated M1 and WT viruses. Samples were analyzed at 16 hours post infection with triplicate samples taken. The mean and standard error within a single experiment are plotted. Statistical analysis was performed using an unpaired two tailed t-test (WT versus M1). P

    Journal: PLoS Pathogens

    Article Title: Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4

    doi: 10.1371/journal.ppat.1002413

    Figure Lengend Snippet: Viruses lacking VF1 are altered in their ability to induce apoptosis. (A) Time course of caspase 3/7 activity in RAW264.7 cells treated with Staurosporine or infected with wild-type MNV-1 (WT) or a VF1 knockout virus (M1). Cells were infected at a MOI of 5 TCID 50 per cell and samples harvested at various times post infection. Caspase 3/7 activity was determined using a commercial assay from Promega (Glo 3/7) and the activity represented as relative light units (RLU). Each assay was performed in triplicate with the average and standard error within a single experiment plotted. Statistical analysis was performed using 2 way ANOVA with Bonfferoni post tests (B) Western blot analysis of extracts prepared from cells infected as detailed in (A). Samples were prepared at various times post infection, separated by SDS-PAGE and subsequently immune blotted using antisera to GAPDH, NS7, VP1 or VF1. Non-infected (Mock) cells were included as controls. (C). Caspase 3/7 activity assay in RAW 264.7 cells infected with M1 or WT MNV-1 or UV-inactivated M1 and WT viruses. Samples were analyzed at 16 hours post infection with triplicate samples taken. The mean and standard error within a single experiment are plotted. Statistical analysis was performed using an unpaired two tailed t-test (WT versus M1). P

    Article Snippet: GFP fusions of VF1 were generated by cloning the VF1 encoding sequence into the GFP plasmids pEGFP-N1 and pEGFP-C1 (Clontech).

    Techniques: Activity Assay, Infection, Knock-Out, Western Blot, SDS Page, Two Tailed Test

    VF1 is not required for murine norovirus replication in tissue culture although there is a ‘fitness cost’ to the virus. (A) Schematic representation of the position and nucleotide changes introduced to generate the VF1 mutant viruses M1, M10 and M20. Nucleotide numbers refer to the positions in the MNV-1 genome. The effect of the introduced nucleotide changes on the VF1 and VP1 protein sequences are also illustrated. Note that the mutations introduced in M1, M10 and M20 do not affect the coding sequence of the capsid protein VP1 but introduce a stop codon into VF1 at various positions. (B) Coupled in vitro transcription and translation reactions confirming the lack of VF1 expression in M1 and M10 viruses, with M20 producing a marginally shorter VF1 product. PCR products containing the subgenomic RNA region under control of a T7 RNA polymerase promoter were generated from cDNA constructs of either wild-type (WT), M1, M10 or M20 VF1 mutants and subsequently used for transcription and translation (TNT) in vitro in the presence of S35 methionine. Radioactively labeled proteins were subsequent resolved by SDS-PAGE, prior to exposure to film. VP1* represents a potential shorter VP1 product generated by translation initiation from an AUG initiation codon in-frame yet downstream from the authentic VP1 AUG. (C) Western blot analysis of RAW264.7 cells infected with low passage, sequence verified stocks of either wild-type (WT), M1, M10 or M20 VF1 mutant viruses. RAW264.7 cells were infected at a MOI 10 TCID 50 per cell and harvested 12 hours post infection, prior to separation by SDS-PAGE and western blot using either anti-VF1 or anti-VP2 antisera. (D) Multi-cycle growth kinetics analysis of VF1 mutant viruses M1, M10 and M20 in RAW264.7 cells. Cells were infected with a MOI of 0.01 TCID 50 per cell and samples harvested at various times post infection prior to titration on RAW264.7 cells. Virus yield is expressed as TCID 50 /ml. Infections were performed in triplicate, with the average virus titer and standard deviation plotted. (E) Sequence chromatograms of M1, M10 and M20 VF1 mutant viruses after passage 1 or 5 in RAW264.7 cells. Viruses obtained from passage 1 and 5 low multiplicity infections (MOI) of RAW264.7 cells were used to infect a subsequent monolayer at high MOI prior to RNA isolation, RT-PCR amplification of the region encompassing ORF4 and sequence analysis. The positions of the introduced stop codons in the mutants M1, M10 and M20 are boxed as are the sequences after 5 repeated passages in cell culture. (F) Western blot analysis of VF1 and VP2 expression in cells infected with either wild-type MNV or passage 5 VF1 mutant viruses M1, M10 and M20. 18 hours post infection at a high MOI (4 TCID 50 per cell) cells were harvested, separated by SDS-PAGE prior to western blot analysis using antisera to either VF1 or VP2. Note that batch-to-batch variation in the quality of the anti-VP2 antisera accounts for the variations in the levels of non-specific proteins detected in panels C and F.

    Journal: PLoS Pathogens

    Article Title: Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4

    doi: 10.1371/journal.ppat.1002413

    Figure Lengend Snippet: VF1 is not required for murine norovirus replication in tissue culture although there is a ‘fitness cost’ to the virus. (A) Schematic representation of the position and nucleotide changes introduced to generate the VF1 mutant viruses M1, M10 and M20. Nucleotide numbers refer to the positions in the MNV-1 genome. The effect of the introduced nucleotide changes on the VF1 and VP1 protein sequences are also illustrated. Note that the mutations introduced in M1, M10 and M20 do not affect the coding sequence of the capsid protein VP1 but introduce a stop codon into VF1 at various positions. (B) Coupled in vitro transcription and translation reactions confirming the lack of VF1 expression in M1 and M10 viruses, with M20 producing a marginally shorter VF1 product. PCR products containing the subgenomic RNA region under control of a T7 RNA polymerase promoter were generated from cDNA constructs of either wild-type (WT), M1, M10 or M20 VF1 mutants and subsequently used for transcription and translation (TNT) in vitro in the presence of S35 methionine. Radioactively labeled proteins were subsequent resolved by SDS-PAGE, prior to exposure to film. VP1* represents a potential shorter VP1 product generated by translation initiation from an AUG initiation codon in-frame yet downstream from the authentic VP1 AUG. (C) Western blot analysis of RAW264.7 cells infected with low passage, sequence verified stocks of either wild-type (WT), M1, M10 or M20 VF1 mutant viruses. RAW264.7 cells were infected at a MOI 10 TCID 50 per cell and harvested 12 hours post infection, prior to separation by SDS-PAGE and western blot using either anti-VF1 or anti-VP2 antisera. (D) Multi-cycle growth kinetics analysis of VF1 mutant viruses M1, M10 and M20 in RAW264.7 cells. Cells were infected with a MOI of 0.01 TCID 50 per cell and samples harvested at various times post infection prior to titration on RAW264.7 cells. Virus yield is expressed as TCID 50 /ml. Infections were performed in triplicate, with the average virus titer and standard deviation plotted. (E) Sequence chromatograms of M1, M10 and M20 VF1 mutant viruses after passage 1 or 5 in RAW264.7 cells. Viruses obtained from passage 1 and 5 low multiplicity infections (MOI) of RAW264.7 cells were used to infect a subsequent monolayer at high MOI prior to RNA isolation, RT-PCR amplification of the region encompassing ORF4 and sequence analysis. The positions of the introduced stop codons in the mutants M1, M10 and M20 are boxed as are the sequences after 5 repeated passages in cell culture. (F) Western blot analysis of VF1 and VP2 expression in cells infected with either wild-type MNV or passage 5 VF1 mutant viruses M1, M10 and M20. 18 hours post infection at a high MOI (4 TCID 50 per cell) cells were harvested, separated by SDS-PAGE prior to western blot analysis using antisera to either VF1 or VP2. Note that batch-to-batch variation in the quality of the anti-VP2 antisera accounts for the variations in the levels of non-specific proteins detected in panels C and F.

    Article Snippet: GFP fusions of VF1 were generated by cloning the VF1 encoding sequence into the GFP plasmids pEGFP-N1 and pEGFP-C1 (Clontech).

    Techniques: Mutagenesis, Sequencing, Introduce, In Vitro, Expressing, Polymerase Chain Reaction, Generated, Construct, Labeling, SDS Page, Western Blot, Infection, Titration, Standard Deviation, Isolation, Reverse Transcription Polymerase Chain Reaction, Amplification, Cell Culture

    Pathology is markedly reduced in the absence of VF1 expression. Age and sex matched STAT1-/- mice were inoculated by oral gavage with 1000 TCID 50 of low passage, sequence verified, wild-type (WT-v) or VF1 knockout (M1-v) viruses generated using a virulent backbone cDNA construct. Necropsies were performed 5 days post infection. Spleen, small intestine and liver tissues were fixed in Bouin's solution, embedded in paraffin wax and sections stained with haematoxylin and eosin.

    Journal: PLoS Pathogens

    Article Title: Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4

    doi: 10.1371/journal.ppat.1002413

    Figure Lengend Snippet: Pathology is markedly reduced in the absence of VF1 expression. Age and sex matched STAT1-/- mice were inoculated by oral gavage with 1000 TCID 50 of low passage, sequence verified, wild-type (WT-v) or VF1 knockout (M1-v) viruses generated using a virulent backbone cDNA construct. Necropsies were performed 5 days post infection. Spleen, small intestine and liver tissues were fixed in Bouin's solution, embedded in paraffin wax and sections stained with haematoxylin and eosin.

    Article Snippet: GFP fusions of VF1 were generated by cloning the VF1 encoding sequence into the GFP plasmids pEGFP-N1 and pEGFP-C1 (Clontech).

    Techniques: Expressing, Mouse Assay, Sequencing, Knock-Out, Generated, Construct, Infection, Staining

    In vitro synthesis of the murine norovirus VF1 protein. (A) Coupled in vitro transcription and translation of a cDNA construct containing the murine norovirus 1 (MNV) sgRNA was performed prior to immunoprecipitation (IP) with polyclonal antisera to VP1, VP2 or VF1. Immunoprecipitated complexes were subsequently resolved by SDS-PAGE (15% polyacrylamide) alongside an aliquot of the complete reaction prior to immunoprecipitation (SG). (B) Western blot analysis indicating VF1 expression during virus high multiplicity infection. RAW264.7 cells were infected with MNV-1 at a MOI of 5 TCID 50 per cell. Protein samples were prepared at various times post infections (HPI) and then separated by SDS-PAGE (15% polyacrylamide) prior to immune-blotting with antisera to VF1, NS7 or VP2.

    Journal: PLoS Pathogens

    Article Title: Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4

    doi: 10.1371/journal.ppat.1002413

    Figure Lengend Snippet: In vitro synthesis of the murine norovirus VF1 protein. (A) Coupled in vitro transcription and translation of a cDNA construct containing the murine norovirus 1 (MNV) sgRNA was performed prior to immunoprecipitation (IP) with polyclonal antisera to VP1, VP2 or VF1. Immunoprecipitated complexes were subsequently resolved by SDS-PAGE (15% polyacrylamide) alongside an aliquot of the complete reaction prior to immunoprecipitation (SG). (B) Western blot analysis indicating VF1 expression during virus high multiplicity infection. RAW264.7 cells were infected with MNV-1 at a MOI of 5 TCID 50 per cell. Protein samples were prepared at various times post infections (HPI) and then separated by SDS-PAGE (15% polyacrylamide) prior to immune-blotting with antisera to VF1, NS7 or VP2.

    Article Snippet: GFP fusions of VF1 were generated by cloning the VF1 encoding sequence into the GFP plasmids pEGFP-N1 and pEGFP-C1 (Clontech).

    Techniques: In Vitro, Construct, Immunoprecipitation, SDS Page, Western Blot, Expressing, Infection

    Viral RNA replication in vivo is reduced as a result of the loss of VF1 expression. Quantitative real-time RT-PCR quantification of viral genome copies in various tissues isolated at 3 (A) or 5 (B) days post infections with either wild type (WT-v) or VF1 knockout (M1-v). At various times post inoculation, tissue samples were isolated and RNA extracted. The genome equivalent per µg of RNA was then determined by comparison to a standard curve generated from in vitro transcribed RNA. The blue line indicates the limit of the detection, based on the ability to detect viral RNA specifically in RNA samples prepared from mock infected animals. Each RNA sample was analyzed at least in duplicate and the mean taken. The mean of each group and standard error is plotted. MLN refers to the mesenteric lymph nodes.

    Journal: PLoS Pathogens

    Article Title: Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4

    doi: 10.1371/journal.ppat.1002413

    Figure Lengend Snippet: Viral RNA replication in vivo is reduced as a result of the loss of VF1 expression. Quantitative real-time RT-PCR quantification of viral genome copies in various tissues isolated at 3 (A) or 5 (B) days post infections with either wild type (WT-v) or VF1 knockout (M1-v). At various times post inoculation, tissue samples were isolated and RNA extracted. The genome equivalent per µg of RNA was then determined by comparison to a standard curve generated from in vitro transcribed RNA. The blue line indicates the limit of the detection, based on the ability to detect viral RNA specifically in RNA samples prepared from mock infected animals. Each RNA sample was analyzed at least in duplicate and the mean taken. The mean of each group and standard error is plotted. MLN refers to the mesenteric lymph nodes.

    Article Snippet: GFP fusions of VF1 were generated by cloning the VF1 encoding sequence into the GFP plasmids pEGFP-N1 and pEGFP-C1 (Clontech).

    Techniques: In Vivo, Expressing, Quantitative RT-PCR, Isolation, Knock-Out, Generated, In Vitro, Infection

    VF1 plays a role in viral virulence. Age and sex matched STAT1-/- mice were inoculated by oral gavage with 1000 TCID 50 of low passage, sequence verified, wild-type (WT-v) or VF1 knockout (M1-v) viruses generated using a virulent backbone cDNA construct. As a measure of the severity of clinical disease, body weight was measured on a daily basis and expressed as percentage of the weight on day 0 prior to inoculation. Mock infected animals were orally inoculated with a control lysate preparation, generated as described in the materials and methods . For clarity the mean weight and the standard error are plotted as both a line graph covering the duration of the experiment (A) and as individual animal weights during days 3-6 (B). Statistical analysis was performed using two-way ANOVA with Bonferroni post-tests (WT-v versus M1-v). The hash and asterisk highlight animal number 776 and 751 used in the sequence analysis on days 5 and 7 respectively as described in the text. Note that animal 751 was removed from the study on day 7 (not shown on the plot) due to humane endpoints.

    Journal: PLoS Pathogens

    Article Title: Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4

    doi: 10.1371/journal.ppat.1002413

    Figure Lengend Snippet: VF1 plays a role in viral virulence. Age and sex matched STAT1-/- mice were inoculated by oral gavage with 1000 TCID 50 of low passage, sequence verified, wild-type (WT-v) or VF1 knockout (M1-v) viruses generated using a virulent backbone cDNA construct. As a measure of the severity of clinical disease, body weight was measured on a daily basis and expressed as percentage of the weight on day 0 prior to inoculation. Mock infected animals were orally inoculated with a control lysate preparation, generated as described in the materials and methods . For clarity the mean weight and the standard error are plotted as both a line graph covering the duration of the experiment (A) and as individual animal weights during days 3-6 (B). Statistical analysis was performed using two-way ANOVA with Bonferroni post-tests (WT-v versus M1-v). The hash and asterisk highlight animal number 776 and 751 used in the sequence analysis on days 5 and 7 respectively as described in the text. Note that animal 751 was removed from the study on day 7 (not shown on the plot) due to humane endpoints.

    Article Snippet: GFP fusions of VF1 were generated by cloning the VF1 encoding sequence into the GFP plasmids pEGFP-N1 and pEGFP-C1 (Clontech).

    Techniques: Mouse Assay, Sequencing, Knock-Out, Generated, Construct, Infection

    VF1 expression is required for virus efficient replication in vivo . Age and sex matched C57BL/6 mice were inoculated by oral gavage with 1×10 7 TCID 50 of low passage, sequence verified, wild-type (WT) or VF1 knockout (M1) viruses. Body weight was measured on a daily basis and expressed as a percentage of the weight on day 0 prior to inoculation. The mean weight and the standard error are plotted as a line graph covering the duration of the experiment (A). Quantitative real-time RT-PCR quantification of viral genome copies in the mesenteric lymph node isolated from mice at various times post inoculation (B). The mean and standard error are plotted. Statistical analysis was performed using two-way ANOVA and Bonferroni post tests (WT versus M1). The blue line represents the detection limit as determined by the sensitivity of qPCR to detect standards, equating to 80 copies per µg of RNA. Samples that were below the detection limit were set as 80 copies per µg of RNA.

    Journal: PLoS Pathogens

    Article Title: Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4

    doi: 10.1371/journal.ppat.1002413

    Figure Lengend Snippet: VF1 expression is required for virus efficient replication in vivo . Age and sex matched C57BL/6 mice were inoculated by oral gavage with 1×10 7 TCID 50 of low passage, sequence verified, wild-type (WT) or VF1 knockout (M1) viruses. Body weight was measured on a daily basis and expressed as a percentage of the weight on day 0 prior to inoculation. The mean weight and the standard error are plotted as a line graph covering the duration of the experiment (A). Quantitative real-time RT-PCR quantification of viral genome copies in the mesenteric lymph node isolated from mice at various times post inoculation (B). The mean and standard error are plotted. Statistical analysis was performed using two-way ANOVA and Bonferroni post tests (WT versus M1). The blue line represents the detection limit as determined by the sensitivity of qPCR to detect standards, equating to 80 copies per µg of RNA. Samples that were below the detection limit were set as 80 copies per µg of RNA.

    Article Snippet: GFP fusions of VF1 were generated by cloning the VF1 encoding sequence into the GFP plasmids pEGFP-N1 and pEGFP-C1 (Clontech).

    Techniques: Expressing, In Vivo, Mouse Assay, Sequencing, Knock-Out, Quantitative RT-PCR, Isolation, Real-time Polymerase Chain Reaction

    VF1 localizes to mitochondria. (A) Confocal microscopy of cells transfected with either N- or C-terminal fusions of EGFP to VF1. Cos7 cells were transfected with plasmids expressing VF1-EGFP fusion proteins, prior to co-staining with Mitotracker, fixation and staining with DAPI. Cells were analyzed using a Zeiss Meta510 confocal microscope. GFP transfected and Mitotracker stained Cos7 cells are also shown as a control. (B) Biochemical fractionation demonstrates that VF1 localizes to mitochondria during virus infection. RAW264.7 cells were either mock infected or infected with wild-type MNV followed by mitochondrial purification 12 hours post infection. Samples were subsequently analyzed by western blot using antisera to VF1, apoptosis inducing factor 1 (AIF) or poly rC-binding protein1/2 (PCBP). Total cellular lysate was prepared by lysing cells directly in SDS-PAGE sample buffer and run alongside as a control.

    Journal: PLoS Pathogens

    Article Title: Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4

    doi: 10.1371/journal.ppat.1002413

    Figure Lengend Snippet: VF1 localizes to mitochondria. (A) Confocal microscopy of cells transfected with either N- or C-terminal fusions of EGFP to VF1. Cos7 cells were transfected with plasmids expressing VF1-EGFP fusion proteins, prior to co-staining with Mitotracker, fixation and staining with DAPI. Cells were analyzed using a Zeiss Meta510 confocal microscope. GFP transfected and Mitotracker stained Cos7 cells are also shown as a control. (B) Biochemical fractionation demonstrates that VF1 localizes to mitochondria during virus infection. RAW264.7 cells were either mock infected or infected with wild-type MNV followed by mitochondrial purification 12 hours post infection. Samples were subsequently analyzed by western blot using antisera to VF1, apoptosis inducing factor 1 (AIF) or poly rC-binding protein1/2 (PCBP). Total cellular lysate was prepared by lysing cells directly in SDS-PAGE sample buffer and run alongside as a control.

    Article Snippet: GFP fusions of VF1 were generated by cloning the VF1 encoding sequence into the GFP plasmids pEGFP-N1 and pEGFP-C1 (Clontech).

    Techniques: Confocal Microscopy, Transfection, Expressing, Staining, Microscopy, Fractionation, Infection, Purification, Western Blot, Binding Assay, SDS Page

    The Change in Chain Length Distribution of Amylopectin in Rice Endosperm between Various pho1 Mutants and Their Wild-Type Parents (T65 or Kinmaze) as Determined by the APTS-Capillary Electrophoresis Method.

    Journal:

    Article Title: Mutation of the Plastidial ?-Glucan Phosphorylase Gene in Rice Affects the Synthesis and Structure of Starch in the Endosperm

    doi: 10.1105/tpc.107.054007

    Figure Lengend Snippet: The Change in Chain Length Distribution of Amylopectin in Rice Endosperm between Various pho1 Mutants and Their Wild-Type Parents (T65 or Kinmaze) as Determined by the APTS-Capillary Electrophoresis Method.

    Article Snippet: Liquid chromatography–tandem mass spectrometry analysis of the tryptic peptide fragments derived from the 106-kD protein showed that the peptide sequences aligned with the rice Pho1 primary sequence consisting of 928 amino acids (National Center for Biotechnology Information [NCBI] BLAST of gil13195340) ( ; see Supplemental Figure 1 online).

    Techniques: Electrophoresis

    Subcellular Localization of Pho1 in Developing Endosperm Cells of Rice.

    Journal:

    Article Title: Mutation of the Plastidial ?-Glucan Phosphorylase Gene in Rice Affects the Synthesis and Structure of Starch in the Endosperm

    doi: 10.1105/tpc.107.054007

    Figure Lengend Snippet: Subcellular Localization of Pho1 in Developing Endosperm Cells of Rice.

    Article Snippet: Liquid chromatography–tandem mass spectrometry analysis of the tryptic peptide fragments derived from the 106-kD protein showed that the peptide sequences aligned with the rice Pho1 primary sequence consisting of 928 amino acids (National Center for Biotechnology Information [NCBI] BLAST of gil13195340) ( ; see Supplemental Figure 1 online).

    Techniques:

    Effects of pho1 Mutation on Starch Granules as Viewed by Scanning Electron Microscopy.

    Journal:

    Article Title: Mutation of the Plastidial ?-Glucan Phosphorylase Gene in Rice Affects the Synthesis and Structure of Starch in the Endosperm

    doi: 10.1105/tpc.107.054007

    Figure Lengend Snippet: Effects of pho1 Mutation on Starch Granules as Viewed by Scanning Electron Microscopy.

    Article Snippet: Liquid chromatography–tandem mass spectrometry analysis of the tryptic peptide fragments derived from the 106-kD protein showed that the peptide sequences aligned with the rice Pho1 primary sequence consisting of 928 amino acids (National Center for Biotechnology Information [NCBI] BLAST of gil13195340) ( ; see Supplemental Figure 1 online).

    Techniques: Mutagenesis, Electron Microscopy

    Gene Dosage Effects of pho1 Mutant Gene on the Activity Staining and Protein Gel Blot Band Patterns of Pho1

    Journal:

    Article Title: Mutation of the Plastidial ?-Glucan Phosphorylase Gene in Rice Affects the Synthesis and Structure of Starch in the Endosperm

    doi: 10.1105/tpc.107.054007

    Figure Lengend Snippet: Gene Dosage Effects of pho1 Mutant Gene on the Activity Staining and Protein Gel Blot Band Patterns of Pho1

    Article Snippet: Liquid chromatography–tandem mass spectrometry analysis of the tryptic peptide fragments derived from the 106-kD protein showed that the peptide sequences aligned with the rice Pho1 primary sequence consisting of 928 amino acids (National Center for Biotechnology Information [NCBI] BLAST of gil13195340) ( ; see Supplemental Figure 1 online).

    Techniques: Mutagenesis, Activity Assay, Staining, Western Blot

    Effects of pho1 Mutation on Pho1 Content and Activity in Rice Endosperm and on Morphology of the Kernel.

    Journal:

    Article Title: Mutation of the Plastidial ?-Glucan Phosphorylase Gene in Rice Affects the Synthesis and Structure of Starch in the Endosperm

    doi: 10.1105/tpc.107.054007

    Figure Lengend Snippet: Effects of pho1 Mutation on Pho1 Content and Activity in Rice Endosperm and on Morphology of the Kernel.

    Article Snippet: Liquid chromatography–tandem mass spectrometry analysis of the tryptic peptide fragments derived from the 106-kD protein showed that the peptide sequences aligned with the rice Pho1 primary sequence consisting of 928 amino acids (National Center for Biotechnology Information [NCBI] BLAST of gil13195340) ( ; see Supplemental Figure 1 online).

    Techniques: Mutagenesis, Activity Assay

    Schematic Representation of Mutations in the Pho1 Gene of BMF136 and EM640.

    Journal:

    Article Title: Mutation of the Plastidial ?-Glucan Phosphorylase Gene in Rice Affects the Synthesis and Structure of Starch in the Endosperm

    doi: 10.1105/tpc.107.054007

    Figure Lengend Snippet: Schematic Representation of Mutations in the Pho1 Gene of BMF136 and EM640.

    Article Snippet: Liquid chromatography–tandem mass spectrometry analysis of the tryptic peptide fragments derived from the 106-kD protein showed that the peptide sequences aligned with the rice Pho1 primary sequence consisting of 928 amino acids (National Center for Biotechnology Information [NCBI] BLAST of gil13195340) ( ; see Supplemental Figure 1 online).

    Techniques:

    Genetic Analysis of the pho1 Mutation in Rice.

    Journal:

    Article Title: Mutation of the Plastidial ?-Glucan Phosphorylase Gene in Rice Affects the Synthesis and Structure of Starch in the Endosperm

    doi: 10.1105/tpc.107.054007

    Figure Lengend Snippet: Genetic Analysis of the pho1 Mutation in Rice.

    Article Snippet: Liquid chromatography–tandem mass spectrometry analysis of the tryptic peptide fragments derived from the 106-kD protein showed that the peptide sequences aligned with the rice Pho1 primary sequence consisting of 928 amino acids (National Center for Biotechnology Information [NCBI] BLAST of gil13195340) ( ; see Supplemental Figure 1 online).

    Techniques: Mutagenesis

    Starch Accumulation in the Endosperm of the pho1 Mutant Was Affected by Temperature

    Journal:

    Article Title: Mutation of the Plastidial ?-Glucan Phosphorylase Gene in Rice Affects the Synthesis and Structure of Starch in the Endosperm

    doi: 10.1105/tpc.107.054007

    Figure Lengend Snippet: Starch Accumulation in the Endosperm of the pho1 Mutant Was Affected by Temperature

    Article Snippet: Liquid chromatography–tandem mass spectrometry analysis of the tryptic peptide fragments derived from the 106-kD protein showed that the peptide sequences aligned with the rice Pho1 primary sequence consisting of 928 amino acids (National Center for Biotechnology Information [NCBI] BLAST of gil13195340) ( ; see Supplemental Figure 1 online).

    Techniques: Mutagenesis

    Model Representing the Possible Role of Pho1 in Starch Biosynthesis in the Rice Endosperm.

    Journal:

    Article Title: Mutation of the Plastidial ?-Glucan Phosphorylase Gene in Rice Affects the Synthesis and Structure of Starch in the Endosperm

    doi: 10.1105/tpc.107.054007

    Figure Lengend Snippet: Model Representing the Possible Role of Pho1 in Starch Biosynthesis in the Rice Endosperm.

    Article Snippet: Liquid chromatography–tandem mass spectrometry analysis of the tryptic peptide fragments derived from the 106-kD protein showed that the peptide sequences aligned with the rice Pho1 primary sequence consisting of 928 amino acids (National Center for Biotechnology Information [NCBI] BLAST of gil13195340) ( ; see Supplemental Figure 1 online).

    Techniques:

    Effects of pho1 Mutations on Activities of Starch Biosynthetic Enzymes.

    Journal:

    Article Title: Mutation of the Plastidial ?-Glucan Phosphorylase Gene in Rice Affects the Synthesis and Structure of Starch in the Endosperm

    doi: 10.1105/tpc.107.054007

    Figure Lengend Snippet: Effects of pho1 Mutations on Activities of Starch Biosynthetic Enzymes.

    Article Snippet: Liquid chromatography–tandem mass spectrometry analysis of the tryptic peptide fragments derived from the 106-kD protein showed that the peptide sequences aligned with the rice Pho1 primary sequence consisting of 928 amino acids (National Center for Biotechnology Information [NCBI] BLAST of gil13195340) ( ; see Supplemental Figure 1 online).

    Techniques:

    The pho1 Mutation Changes the Gelatinization Properties of Starch in Rice Endosperm

    Journal:

    Article Title: Mutation of the Plastidial ?-Glucan Phosphorylase Gene in Rice Affects the Synthesis and Structure of Starch in the Endosperm

    doi: 10.1105/tpc.107.054007

    Figure Lengend Snippet: The pho1 Mutation Changes the Gelatinization Properties of Starch in Rice Endosperm

    Article Snippet: Liquid chromatography–tandem mass spectrometry analysis of the tryptic peptide fragments derived from the 106-kD protein showed that the peptide sequences aligned with the rice Pho1 primary sequence consisting of 928 amino acids (National Center for Biotechnology Information [NCBI] BLAST of gil13195340) ( ; see Supplemental Figure 1 online).

    Techniques: Mutagenesis

    Effects of Temperature on Kernel Morphology during Development of pho1 Mutant Seeds.

    Journal:

    Article Title: Mutation of the Plastidial ?-Glucan Phosphorylase Gene in Rice Affects the Synthesis and Structure of Starch in the Endosperm

    doi: 10.1105/tpc.107.054007

    Figure Lengend Snippet: Effects of Temperature on Kernel Morphology during Development of pho1 Mutant Seeds.

    Article Snippet: Liquid chromatography–tandem mass spectrometry analysis of the tryptic peptide fragments derived from the 106-kD protein showed that the peptide sequences aligned with the rice Pho1 primary sequence consisting of 928 amino acids (National Center for Biotechnology Information [NCBI] BLAST of gil13195340) ( ; see Supplemental Figure 1 online).

    Techniques: Mutagenesis

    Cryosubstituted, Cryofixed Postembedding Immunoelectron Microscopy with Anti-CGI-58 Antiserum in Normal Human Epidermis. A: Anti-CGI-58 antibody deposition labeled with 5 nm gold particles was seen on cytoplasmic vesicles ( arrows ) in the uppermost spinous

    Journal:

    Article Title: CGI-58 Is an ?/?-Hydrolase within Lipid Transporting Lamellar Granules of Differentiated Keratinocytes

    doi: 10.2353/ajpath.2008.080005

    Figure Lengend Snippet: Cryosubstituted, Cryofixed Postembedding Immunoelectron Microscopy with Anti-CGI-58 Antiserum in Normal Human Epidermis. A: Anti-CGI-58 antibody deposition labeled with 5 nm gold particles was seen on cytoplasmic vesicles ( arrows ) in the uppermost spinous

    Article Snippet: Polyclonal anti-CGI-58 antibody was raised in rabbits using a 14 amino acid sequence synthetic peptide (residues 181-194) derived from the CGI-58 sequence ( ) as the immunogen (Sigma Genosys, Hokkaido, Japan).

    Techniques: Immuno-Electron Microscopy, Labeling

    CGI-58 Immunolabeling in Harlequin Ichthyosis Patients’ Epidermis. In skin specimens from the two harlequin ichthyosis patients with defective LGs ( A, B ), extremely reduced CGI-58 expression was seen compared with strong CGI-58 staining in normal

    Journal:

    Article Title: CGI-58 Is an ?/?-Hydrolase within Lipid Transporting Lamellar Granules of Differentiated Keratinocytes

    doi: 10.2353/ajpath.2008.080005

    Figure Lengend Snippet: CGI-58 Immunolabeling in Harlequin Ichthyosis Patients’ Epidermis. In skin specimens from the two harlequin ichthyosis patients with defective LGs ( A, B ), extremely reduced CGI-58 expression was seen compared with strong CGI-58 staining in normal

    Article Snippet: Polyclonal anti-CGI-58 antibody was raised in rabbits using a 14 amino acid sequence synthetic peptide (residues 181-194) derived from the CGI-58 sequence ( ) as the immunogen (Sigma Genosys, Hokkaido, Japan).

    Techniques: Immunolabeling, Expressing, Staining

    CGI-58 Expression during Human Keratinocyte Differentiation. A: Real-time RT-PCR analysis revealed that CGI-58 mRNA expression in human keratinocytes cultured in the high Ca 2+ condition was 2.3-fold higher than that of cultured keratinocytes in

    Journal:

    Article Title: CGI-58 Is an ?/?-Hydrolase within Lipid Transporting Lamellar Granules of Differentiated Keratinocytes

    doi: 10.2353/ajpath.2008.080005

    Figure Lengend Snippet: CGI-58 Expression during Human Keratinocyte Differentiation. A: Real-time RT-PCR analysis revealed that CGI-58 mRNA expression in human keratinocytes cultured in the high Ca 2+ condition was 2.3-fold higher than that of cultured keratinocytes in

    Article Snippet: Polyclonal anti-CGI-58 antibody was raised in rabbits using a 14 amino acid sequence synthetic peptide (residues 181-194) derived from the CGI-58 sequence ( ) as the immunogen (Sigma Genosys, Hokkaido, Japan).

    Techniques: Expressing, Quantitative RT-PCR, Cell Culture

    CGI-58 Distribution in the Skin. A, B: Immunoreactivity of the anti-CGI-58 antiserum in normal human skin. CGI-58 immunolabeling (green, FITC) was seen in the epidermis, predominantly in the granular layer ( arrows ) and in the dermal fibroblasts in human

    Journal:

    Article Title: CGI-58 Is an ?/?-Hydrolase within Lipid Transporting Lamellar Granules of Differentiated Keratinocytes

    doi: 10.2353/ajpath.2008.080005

    Figure Lengend Snippet: CGI-58 Distribution in the Skin. A, B: Immunoreactivity of the anti-CGI-58 antiserum in normal human skin. CGI-58 immunolabeling (green, FITC) was seen in the epidermis, predominantly in the granular layer ( arrows ) and in the dermal fibroblasts in human

    Article Snippet: Polyclonal anti-CGI-58 antibody was raised in rabbits using a 14 amino acid sequence synthetic peptide (residues 181-194) derived from the CGI-58 sequence ( ) as the immunogen (Sigma Genosys, Hokkaido, Japan).

    Techniques: Immunolabeling

    CGI-58 Distribution in the Brain and the Liver. Immunohistochemical localization of CGI-58 in the mouse brain ( A–E ) and liver ( F, G ). A-D: Hypothalamus at lower magnification ( A ) and at higher magnification ( B–E ). A: CGI-58-positive neurons

    Journal:

    Article Title: CGI-58 Is an ?/?-Hydrolase within Lipid Transporting Lamellar Granules of Differentiated Keratinocytes

    doi: 10.2353/ajpath.2008.080005

    Figure Lengend Snippet: CGI-58 Distribution in the Brain and the Liver. Immunohistochemical localization of CGI-58 in the mouse brain ( A–E ) and liver ( F, G ). A-D: Hypothalamus at lower magnification ( A ) and at higher magnification ( B–E ). A: CGI-58-positive neurons

    Article Snippet: Polyclonal anti-CGI-58 antibody was raised in rabbits using a 14 amino acid sequence synthetic peptide (residues 181-194) derived from the CGI-58 sequence ( ) as the immunogen (Sigma Genosys, Hokkaido, Japan).

    Techniques: Immunohistochemistry

    Lentiviral-Mediated Knockdown of CGI-58 Expression in Cultures of Human Keratinocytes. Two lentiviruses targeting different regions of CGI-58 (miRNA-CGI-58 #1 and miRNA-CGI-58 #2) showed similar depletion/knockdown of CGI-58 mRNA expression. Expression

    Journal:

    Article Title: CGI-58 Is an ?/?-Hydrolase within Lipid Transporting Lamellar Granules of Differentiated Keratinocytes

    doi: 10.2353/ajpath.2008.080005

    Figure Lengend Snippet: Lentiviral-Mediated Knockdown of CGI-58 Expression in Cultures of Human Keratinocytes. Two lentiviruses targeting different regions of CGI-58 (miRNA-CGI-58 #1 and miRNA-CGI-58 #2) showed similar depletion/knockdown of CGI-58 mRNA expression. Expression

    Article Snippet: Polyclonal anti-CGI-58 antibody was raised in rabbits using a 14 amino acid sequence synthetic peptide (residues 181-194) derived from the CGI-58 sequence ( ) as the immunogen (Sigma Genosys, Hokkaido, Japan).

    Techniques: Expressing

    Expression of CGI-58 in developing human skin ( A ) in two-layered epidermis (at 49 days EGA), weak CGI-58 staining (green, FITC; white arrows ) was seen in the epidermis; ( B ) in the three-layered epidermis (76 days EGA), CGI-58 staining was observed only

    Journal:

    Article Title: CGI-58 Is an ?/?-Hydrolase within Lipid Transporting Lamellar Granules of Differentiated Keratinocytes

    doi: 10.2353/ajpath.2008.080005

    Figure Lengend Snippet: Expression of CGI-58 in developing human skin ( A ) in two-layered epidermis (at 49 days EGA), weak CGI-58 staining (green, FITC; white arrows ) was seen in the epidermis; ( B ) in the three-layered epidermis (76 days EGA), CGI-58 staining was observed only

    Article Snippet: Polyclonal anti-CGI-58 antibody was raised in rabbits using a 14 amino acid sequence synthetic peptide (residues 181-194) derived from the CGI-58 sequence ( ) as the immunogen (Sigma Genosys, Hokkaido, Japan).

    Techniques: Expressing, Staining

    Immunoblot Analysis with Anti-CGI-58 Antiserum. Immunoblot analysis with the anti-CGI-58 antiserum using total protein samples extracted from normal human skin, mouse brain, mouse liver, and mouse skin. An approximate 45 kDa band ( arrow ) was detected

    Journal:

    Article Title: CGI-58 Is an ?/?-Hydrolase within Lipid Transporting Lamellar Granules of Differentiated Keratinocytes

    doi: 10.2353/ajpath.2008.080005

    Figure Lengend Snippet: Immunoblot Analysis with Anti-CGI-58 Antiserum. Immunoblot analysis with the anti-CGI-58 antiserum using total protein samples extracted from normal human skin, mouse brain, mouse liver, and mouse skin. An approximate 45 kDa band ( arrow ) was detected

    Article Snippet: Polyclonal anti-CGI-58 antibody was raised in rabbits using a 14 amino acid sequence synthetic peptide (residues 181-194) derived from the CGI-58 sequence ( ) as the immunogen (Sigma Genosys, Hokkaido, Japan).

    Techniques:

    PvTRAg38 expressed on the CHO-K1 cell surface binds to human erythrocytes. The transfected CHO-K1 cells were incubated with mouse monoclonal antibody DL6 directed against the C terminus of the H. simplex glycoprotein D sequences and then stained with

    Journal:

    Article Title: Interaction of Plasmodium vivax Tryptophan-rich Antigen PvTRAg38 with Band 3 on Human Erythrocyte Surface Facilitates Parasite Growth

    doi: 10.1074/jbc.M115.644906

    Figure Lengend Snippet: PvTRAg38 expressed on the CHO-K1 cell surface binds to human erythrocytes. The transfected CHO-K1 cells were incubated with mouse monoclonal antibody DL6 directed against the C terminus of the H. simplex glycoprotein D sequences and then stained with

    Article Snippet: The 18-amino acid-long nonoverlapping peptides, derived from M-PvTRAg38 sequences, were commercially synthesized (Thermo Fisher Scientific) with or without six histidine residues attached to their C terminus.

    Techniques: Transfection, Incubation, Staining

    Defining the Erythrocyte-binding Domains of PvTRAg38

    Journal:

    Article Title: Interaction of Plasmodium vivax Tryptophan-rich Antigen PvTRAg38 with Band 3 on Human Erythrocyte Surface Facilitates Parasite Growth

    doi: 10.1074/jbc.M115.644906

    Figure Lengend Snippet: Defining the Erythrocyte-binding Domains of PvTRAg38

    Article Snippet: The 18-amino acid-long nonoverlapping peptides, derived from M-PvTRAg38 sequences, were commercially synthesized (Thermo Fisher Scientific) with or without six histidine residues attached to their C terminus.

    Techniques: Binding Assay

    Binding of PvTRAg38 peptide P-4 to Band 3 fragments. A , in solid phase ELISA, the plate was coated with (0.5 μ m ) untagged peptide P-4 and reacted with increasing concentrations (0–2 μ m ) of histidine-tagged Band 3 fragments (B3F1,

    Journal:

    Article Title: Interaction of Plasmodium vivax Tryptophan-rich Antigen PvTRAg38 with Band 3 on Human Erythrocyte Surface Facilitates Parasite Growth

    doi: 10.1074/jbc.M115.644906

    Figure Lengend Snippet: Binding of PvTRAg38 peptide P-4 to Band 3 fragments. A , in solid phase ELISA, the plate was coated with (0.5 μ m ) untagged peptide P-4 and reacted with increasing concentrations (0–2 μ m ) of histidine-tagged Band 3 fragments (B3F1,

    Article Snippet: The 18-amino acid-long nonoverlapping peptides, derived from M-PvTRAg38 sequences, were commercially synthesized (Thermo Fisher Scientific) with or without six histidine residues attached to their C terminus.

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

    Identification of erythrocyte membrane proteins interacting with recombinant PvTRAg38 by pulldown assay. A , erythrocyte membrane proteins extracted with 1% C 12 E 8 were incubated with recombinant GST-tagged PvTRAg38 bound to glutathione-Sepharose 4B column,

    Journal:

    Article Title: Interaction of Plasmodium vivax Tryptophan-rich Antigen PvTRAg38 with Band 3 on Human Erythrocyte Surface Facilitates Parasite Growth

    doi: 10.1074/jbc.M115.644906

    Figure Lengend Snippet: Identification of erythrocyte membrane proteins interacting with recombinant PvTRAg38 by pulldown assay. A , erythrocyte membrane proteins extracted with 1% C 12 E 8 were incubated with recombinant GST-tagged PvTRAg38 bound to glutathione-Sepharose 4B column,

    Article Snippet: The 18-amino acid-long nonoverlapping peptides, derived from M-PvTRAg38 sequences, were commercially synthesized (Thermo Fisher Scientific) with or without six histidine residues attached to their C terminus.

    Techniques: Recombinant, Incubation

    Erythrocyte binding activity of the synthetic peptides derived from middle fragment of PvTRAg38. A , the peptides were tested for their erythrocyte binding activity by Cell-ELISA where the ELISA plate was coated with ∼1 million erythrocytes and

    Journal:

    Article Title: Interaction of Plasmodium vivax Tryptophan-rich Antigen PvTRAg38 with Band 3 on Human Erythrocyte Surface Facilitates Parasite Growth

    doi: 10.1074/jbc.M115.644906

    Figure Lengend Snippet: Erythrocyte binding activity of the synthetic peptides derived from middle fragment of PvTRAg38. A , the peptides were tested for their erythrocyte binding activity by Cell-ELISA where the ELISA plate was coated with ∼1 million erythrocytes and

    Article Snippet: The 18-amino acid-long nonoverlapping peptides, derived from M-PvTRAg38 sequences, were commercially synthesized (Thermo Fisher Scientific) with or without six histidine residues attached to their C terminus.

    Techniques: Binding Assay, Activity Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay

    Binding of PvTRAg38 fragments to human erythrocytes. A , PvTRAg38 encoded by exon 2 was divided in to three (N-terminal, middle, and C-terminal) parts. These DNA fragments were PCR-amplified, cloned, and expressed in E. coli for Cell-ELISA or in mammalian

    Journal:

    Article Title: Interaction of Plasmodium vivax Tryptophan-rich Antigen PvTRAg38 with Band 3 on Human Erythrocyte Surface Facilitates Parasite Growth

    doi: 10.1074/jbc.M115.644906

    Figure Lengend Snippet: Binding of PvTRAg38 fragments to human erythrocytes. A , PvTRAg38 encoded by exon 2 was divided in to three (N-terminal, middle, and C-terminal) parts. These DNA fragments were PCR-amplified, cloned, and expressed in E. coli for Cell-ELISA or in mammalian

    Article Snippet: The 18-amino acid-long nonoverlapping peptides, derived from M-PvTRAg38 sequences, were commercially synthesized (Thermo Fisher Scientific) with or without six histidine residues attached to their C terminus.

    Techniques: Binding Assay, Polymerase Chain Reaction, Amplification, Clone Assay, Enzyme-linked Immunosorbent Assay

    Binding of PvTRAg38 fragments and synthetic peptides to Band 3. A , binding of PvTRAg38 fragments with purified Band 3 by solid phase ELISA. Each well of the plate was coated with 50 n m of Band 3 and then allowed to react with increasing concentrations

    Journal:

    Article Title: Interaction of Plasmodium vivax Tryptophan-rich Antigen PvTRAg38 with Band 3 on Human Erythrocyte Surface Facilitates Parasite Growth

    doi: 10.1074/jbc.M115.644906

    Figure Lengend Snippet: Binding of PvTRAg38 fragments and synthetic peptides to Band 3. A , binding of PvTRAg38 fragments with purified Band 3 by solid phase ELISA. Each well of the plate was coated with 50 n m of Band 3 and then allowed to react with increasing concentrations

    Article Snippet: The 18-amino acid-long nonoverlapping peptides, derived from M-PvTRAg38 sequences, were commercially synthesized (Thermo Fisher Scientific) with or without six histidine residues attached to their C terminus.

    Techniques: Binding Assay, Purification, Enzyme-linked Immunosorbent Assay

    Inhibition of P. falciparum growth by PvTRAg38. Parasite culture at late schizont stage was incubated with PvTRAg38, PvTRAg53.7, PfTryThrA, and other controls. After 40 h, parasitemia was determined by ethidium bromide staining measured by flow cytometry

    Journal:

    Article Title: Interaction of Plasmodium vivax Tryptophan-rich Antigen PvTRAg38 with Band 3 on Human Erythrocyte Surface Facilitates Parasite Growth

    doi: 10.1074/jbc.M115.644906

    Figure Lengend Snippet: Inhibition of P. falciparum growth by PvTRAg38. Parasite culture at late schizont stage was incubated with PvTRAg38, PvTRAg53.7, PfTryThrA, and other controls. After 40 h, parasitemia was determined by ethidium bromide staining measured by flow cytometry

    Article Snippet: The 18-amino acid-long nonoverlapping peptides, derived from M-PvTRAg38 sequences, were commercially synthesized (Thermo Fisher Scientific) with or without six histidine residues attached to their C terminus.

    Techniques: Inhibition, Incubation, Staining, Flow Cytometry, Cytometry

    Binding of Band 3 fragments with PvTRAg38. A , solid phase ELISA. The plate was coated with fixed concentration (0.5 μ m ) of untagged PvTRAg38 and reacted with increasing concentrations (0–2 μ m ) of histidine-tagged recombinant Band

    Journal:

    Article Title: Interaction of Plasmodium vivax Tryptophan-rich Antigen PvTRAg38 with Band 3 on Human Erythrocyte Surface Facilitates Parasite Growth

    doi: 10.1074/jbc.M115.644906

    Figure Lengend Snippet: Binding of Band 3 fragments with PvTRAg38. A , solid phase ELISA. The plate was coated with fixed concentration (0.5 μ m ) of untagged PvTRAg38 and reacted with increasing concentrations (0–2 μ m ) of histidine-tagged recombinant Band

    Article Snippet: The 18-amino acid-long nonoverlapping peptides, derived from M-PvTRAg38 sequences, were commercially synthesized (Thermo Fisher Scientific) with or without six histidine residues attached to their C terminus.

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Recombinant

    Binding of PvTRAg38 with purified Band 3 protein. A , Band 3 was purified from human erythrocytes using the protocol of Casey et al. ) by affinity chromatography on aminoethyl-agarose resin. RBC membrane preparations and purified Band 3 were analyzed

    Journal:

    Article Title: Interaction of Plasmodium vivax Tryptophan-rich Antigen PvTRAg38 with Band 3 on Human Erythrocyte Surface Facilitates Parasite Growth

    doi: 10.1074/jbc.M115.644906

    Figure Lengend Snippet: Binding of PvTRAg38 with purified Band 3 protein. A , Band 3 was purified from human erythrocytes using the protocol of Casey et al. ) by affinity chromatography on aminoethyl-agarose resin. RBC membrane preparations and purified Band 3 were analyzed

    Article Snippet: The 18-amino acid-long nonoverlapping peptides, derived from M-PvTRAg38 sequences, were commercially synthesized (Thermo Fisher Scientific) with or without six histidine residues attached to their C terminus.

    Techniques: Binding Assay, Purification, Affinity Chromatography

    Erythrocyte binding activity of PvTRAg38 and its peptides by flow cytometry. A , representative dot plots for binding of PvTRAg38 and its fragments/peptides to human erythrocytes. Normal human RBCs (∼1 million) were incubated with 1 μ m

    Journal:

    Article Title: Interaction of Plasmodium vivax Tryptophan-rich Antigen PvTRAg38 with Band 3 on Human Erythrocyte Surface Facilitates Parasite Growth

    doi: 10.1074/jbc.M115.644906

    Figure Lengend Snippet: Erythrocyte binding activity of PvTRAg38 and its peptides by flow cytometry. A , representative dot plots for binding of PvTRAg38 and its fragments/peptides to human erythrocytes. Normal human RBCs (∼1 million) were incubated with 1 μ m

    Article Snippet: The 18-amino acid-long nonoverlapping peptides, derived from M-PvTRAg38 sequences, were commercially synthesized (Thermo Fisher Scientific) with or without six histidine residues attached to their C terminus.

    Techniques: Binding Assay, Activity Assay, Flow Cytometry, Cytometry, Incubation

    Molecular dynamics of Coq6p homology models. A): Time-evolution of substrate access channel diameter at choke points over MD runs for Coq6p_MODELLER homology model: wild-type (blue trace), Coq6p-G248R (red trace) and Coq6p-L382E (green trace) single mutants, and the Coq6p-G248R-L382E double mutant (purple trace). B): In the wild-type, the channel diameter is large and is constitutively open. C-D): The single mutants display consistently reduced channel diameters which show thermal fluctuations around open and closed states. E): The double mutant displays a channel diameter that is always below the minimum required for substrate binding. In this figure, re face tunnel 1 (purple volume) is oriented directly to the viewer. The pair of residues tracked of the MD calculations as bottlenecks of the channel are represented in sticks.

    Journal: PLoS Computational Biology

    Article Title: Coenzyme Q Biosynthesis: Evidence for a Substrate Access Channel in the FAD-Dependent Monooxygenase Coq6

    doi: 10.1371/journal.pcbi.1004690

    Figure Lengend Snippet: Molecular dynamics of Coq6p homology models. A): Time-evolution of substrate access channel diameter at choke points over MD runs for Coq6p_MODELLER homology model: wild-type (blue trace), Coq6p-G248R (red trace) and Coq6p-L382E (green trace) single mutants, and the Coq6p-G248R-L382E double mutant (purple trace). B): In the wild-type, the channel diameter is large and is constitutively open. C-D): The single mutants display consistently reduced channel diameters which show thermal fluctuations around open and closed states. E): The double mutant displays a channel diameter that is always below the minimum required for substrate binding. In this figure, re face tunnel 1 (purple volume) is oriented directly to the viewer. The pair of residues tracked of the MD calculations as bottlenecks of the channel are represented in sticks.

    Article Snippet: [ ] This process was facilitated by a reference multiple sequence alignment (MSA) of 119 Coq6p sequence homologs obtained by using the ConSurf server.

    Techniques: Mutagenesis, Binding Assay

    Coq6p biochemical characterization. A) SDS PAGE (4–12% Bis-Tris, MOPS). Lane 1, Molecular Weights (kDa); lane 2, BL21 DE3 cells transformed with pMALc2X-Coq6 before IPTG induction; lane 3, cells after 20 hr-IPTG induction at 18°C; lane 4, Coq6p-MBP (96.3 kDa) after a two-step purification (MBP trap, Superdex200 26/60). B) UV-visible spectrum of purified Coq6p-MBP, 1.20 mg/ml.

    Journal: PLoS Computational Biology

    Article Title: Coenzyme Q Biosynthesis: Evidence for a Substrate Access Channel in the FAD-Dependent Monooxygenase Coq6

    doi: 10.1371/journal.pcbi.1004690

    Figure Lengend Snippet: Coq6p biochemical characterization. A) SDS PAGE (4–12% Bis-Tris, MOPS). Lane 1, Molecular Weights (kDa); lane 2, BL21 DE3 cells transformed with pMALc2X-Coq6 before IPTG induction; lane 3, cells after 20 hr-IPTG induction at 18°C; lane 4, Coq6p-MBP (96.3 kDa) after a two-step purification (MBP trap, Superdex200 26/60). B) UV-visible spectrum of purified Coq6p-MBP, 1.20 mg/ml.

    Article Snippet: [ ] This process was facilitated by a reference multiple sequence alignment (MSA) of 119 Coq6p sequence homologs obtained by using the ConSurf server.

    Techniques: SDS Page, Transformation Assay, Purification

    A) Evolutionary residue conservation in Coq6p as calculated by the ConSurf method [ 30 , 31 ] and projected onto the manual Coq6p homology model, space-filling rendering. The conservation scores can identify the FAD binding site (not visible in this view). B) Residue conservation scores also identify a group of conserved residues along the re face tunnel 1 (represented in magenta) reaching the surface and forming a depression (delineated by a black circle in panel A). This surface depression leads to a void volume contiguous with the catalytic site, and is lined with highly conserved residues represented in magenta sticks. For clarity, only the residues present at the tunnel entrance are shown. This set of residues is generally hydrophobic, with rare exception, such as S265. The polar residues may participate in contacting the substrate’s aromatic head as it traverses the channel to the catalytic site. FAD is represented in green stick.

    Journal: PLoS Computational Biology

    Article Title: Coenzyme Q Biosynthesis: Evidence for a Substrate Access Channel in the FAD-Dependent Monooxygenase Coq6

    doi: 10.1371/journal.pcbi.1004690

    Figure Lengend Snippet: A) Evolutionary residue conservation in Coq6p as calculated by the ConSurf method [ 30 , 31 ] and projected onto the manual Coq6p homology model, space-filling rendering. The conservation scores can identify the FAD binding site (not visible in this view). B) Residue conservation scores also identify a group of conserved residues along the re face tunnel 1 (represented in magenta) reaching the surface and forming a depression (delineated by a black circle in panel A). This surface depression leads to a void volume contiguous with the catalytic site, and is lined with highly conserved residues represented in magenta sticks. For clarity, only the residues present at the tunnel entrance are shown. This set of residues is generally hydrophobic, with rare exception, such as S265. The polar residues may participate in contacting the substrate’s aromatic head as it traverses the channel to the catalytic site. FAD is represented in green stick.

    Article Snippet: [ ] This process was facilitated by a reference multiple sequence alignment (MSA) of 119 Coq6p sequence homologs obtained by using the ConSurf server.

    Techniques: Binding Assay

    Volume rendering of the channels system identified in the resting Coq6p enzyme models: (A) Coq6p_I-TASSER model, (B) Coq6p_ROBETTA model and (C) Coq6p_MODELLER model. Three channels are identified that converge on the catalytic site close to the FAD isoalloxazine (in green stick). Two of these tunnels (1 and 2, in purple and blue, respectively) exit the enzyme from the re face, the other (3, in red) from the si face of the isoalloxazine ring. Bottleneck residues used in MD channel diameter tracking are represented as sticks.

    Journal: PLoS Computational Biology

    Article Title: Coenzyme Q Biosynthesis: Evidence for a Substrate Access Channel in the FAD-Dependent Monooxygenase Coq6

    doi: 10.1371/journal.pcbi.1004690

    Figure Lengend Snippet: Volume rendering of the channels system identified in the resting Coq6p enzyme models: (A) Coq6p_I-TASSER model, (B) Coq6p_ROBETTA model and (C) Coq6p_MODELLER model. Three channels are identified that converge on the catalytic site close to the FAD isoalloxazine (in green stick). Two of these tunnels (1 and 2, in purple and blue, respectively) exit the enzyme from the re face, the other (3, in red) from the si face of the isoalloxazine ring. Bottleneck residues used in MD channel diameter tracking are represented as sticks.

    Article Snippet: [ ] This process was facilitated by a reference multiple sequence alignment (MSA) of 119 Coq6p sequence homologs obtained by using the ConSurf server.

    Techniques:

    Comparison of the three Coq6p homology models. The Coq6p enzyme secondary structure is shown in cartoon, while the FAD cofactor is shown in green stick. Structurally divergent Coq6p secondary structure elements are highlighted in orange (the C-terminus) and purple (the Coq6-family insert). From left to right: Coq6p_I-TASSER model, Coq6p_ROBETTA model, and the Coq6p_MODELLER model. The isoalloxazine ring plane of the FAD co-factor has two faces, named si and re , with the relevant stereocenter for naming the faces being the C2’ carbon of the ribityl chain. Each face of the FAD’s ring is used here to designate the half of the Coq6p enzyme extending from the si and re faces of the FAD binding pocket to the edge of the protein. The top row shows the models from the re face, while the bottom row is rotated 90 degrees about the vertical to show the exterior of the beta sheet domain, which bears the insert.

    Journal: PLoS Computational Biology

    Article Title: Coenzyme Q Biosynthesis: Evidence for a Substrate Access Channel in the FAD-Dependent Monooxygenase Coq6

    doi: 10.1371/journal.pcbi.1004690

    Figure Lengend Snippet: Comparison of the three Coq6p homology models. The Coq6p enzyme secondary structure is shown in cartoon, while the FAD cofactor is shown in green stick. Structurally divergent Coq6p secondary structure elements are highlighted in orange (the C-terminus) and purple (the Coq6-family insert). From left to right: Coq6p_I-TASSER model, Coq6p_ROBETTA model, and the Coq6p_MODELLER model. The isoalloxazine ring plane of the FAD co-factor has two faces, named si and re , with the relevant stereocenter for naming the faces being the C2’ carbon of the ribityl chain. Each face of the FAD’s ring is used here to designate the half of the Coq6p enzyme extending from the si and re faces of the FAD binding pocket to the edge of the protein. The top row shows the models from the re face, while the bottom row is rotated 90 degrees about the vertical to show the exterior of the beta sheet domain, which bears the insert.

    Article Snippet: [ ] This process was facilitated by a reference multiple sequence alignment (MSA) of 119 Coq6p sequence homologs obtained by using the ConSurf server.

    Techniques: Binding Assay

    A) 10 fold serial dilution of the Δcoq6 strain carrying an empty plasmid (vec) or plasmids coding for Coq6p, Coq6p-G248R, Coq6p-L382E and Coq6p-G248R-L382E. The plates contained YNB-pABA agar medium supplemented with the indicated carbon source and vanillic acid (VA) or not. The plates were imaged after incubation at 30°C for 2 days (glucose) or 6 days (lactate-glycerol). B) Representative electrochromatogram of lipid extracts from Δcoq6 cells expressing either Coq6p, Coq6p-G248R, Coq6p-L382E and Coq6p-G248R-L382E (1 mg of cells). The elution position of the Q 4 standard, of 3-hexaprenyl-4-hydroxyphenol (4-HP6) and Q 6 are indicated. C) Q6 amounts (in pmoles per mg of wet weight) in Δcoq6 cells expressing either Coq6p, Coq6p-G248R, Coq6p-L382E or Coq6p-G248R-L382E. Cells were grown in YNB–pABA 2% lactate-glycerol containing 10 μM 4HB. The results are the average of 3–4 independent experiments and error bars represent standard deviation. ND, not detected.

    Journal: PLoS Computational Biology

    Article Title: Coenzyme Q Biosynthesis: Evidence for a Substrate Access Channel in the FAD-Dependent Monooxygenase Coq6

    doi: 10.1371/journal.pcbi.1004690

    Figure Lengend Snippet: A) 10 fold serial dilution of the Δcoq6 strain carrying an empty plasmid (vec) or plasmids coding for Coq6p, Coq6p-G248R, Coq6p-L382E and Coq6p-G248R-L382E. The plates contained YNB-pABA agar medium supplemented with the indicated carbon source and vanillic acid (VA) or not. The plates were imaged after incubation at 30°C for 2 days (glucose) or 6 days (lactate-glycerol). B) Representative electrochromatogram of lipid extracts from Δcoq6 cells expressing either Coq6p, Coq6p-G248R, Coq6p-L382E and Coq6p-G248R-L382E (1 mg of cells). The elution position of the Q 4 standard, of 3-hexaprenyl-4-hydroxyphenol (4-HP6) and Q 6 are indicated. C) Q6 amounts (in pmoles per mg of wet weight) in Δcoq6 cells expressing either Coq6p, Coq6p-G248R, Coq6p-L382E or Coq6p-G248R-L382E. Cells were grown in YNB–pABA 2% lactate-glycerol containing 10 μM 4HB. The results are the average of 3–4 independent experiments and error bars represent standard deviation. ND, not detected.

    Article Snippet: [ ] This process was facilitated by a reference multiple sequence alignment (MSA) of 119 Coq6p sequence homologs obtained by using the ConSurf server.

    Techniques: Serial Dilution, Plasmid Preparation, Incubation, Expressing, Standard Deviation

    Model of FAD bound to Coq6p, as shown in the Coq6p_MODELLER model. This pose is from a FAD bound conformation selected from MD simulations (C: black, N: blue,O: red, P: orange). Hydrogen bonds are in green dashed lines, and π-type interactions with FAD’s aromatic cycles in purple dashed lines (centre of mass are shown as clear pink spheres). Residues are in sticks (C: green, S: yellow, O: red, N: blue). The adenine ring’s N3 nitrogen is H-bonded to V167. The adenine aromatic system can also form π-sulfur interactions with M62 and π-anion interactions with D201. The ribose hydroxyl is H-bonded to D61. The pyrophosphate is H-bonded to the side chains of R83 and N204, as well as the backbone nitrogen of F203 (side chain omitted for clarity). The ribityl chain is H-bonded to the side-chain of D374, the D of the GDAxH motif. The isoalloxazine ring can form π − π interactions with the F354 side chain (backbone omitted for clarity). These residues are highly conserved in the Coq6 family ( S5 Fig ; highlighted in green in Fig 2 ), with the exception of M62 and F203.

    Journal: PLoS Computational Biology

    Article Title: Coenzyme Q Biosynthesis: Evidence for a Substrate Access Channel in the FAD-Dependent Monooxygenase Coq6

    doi: 10.1371/journal.pcbi.1004690

    Figure Lengend Snippet: Model of FAD bound to Coq6p, as shown in the Coq6p_MODELLER model. This pose is from a FAD bound conformation selected from MD simulations (C: black, N: blue,O: red, P: orange). Hydrogen bonds are in green dashed lines, and π-type interactions with FAD’s aromatic cycles in purple dashed lines (centre of mass are shown as clear pink spheres). Residues are in sticks (C: green, S: yellow, O: red, N: blue). The adenine ring’s N3 nitrogen is H-bonded to V167. The adenine aromatic system can also form π-sulfur interactions with M62 and π-anion interactions with D201. The ribose hydroxyl is H-bonded to D61. The pyrophosphate is H-bonded to the side chains of R83 and N204, as well as the backbone nitrogen of F203 (side chain omitted for clarity). The ribityl chain is H-bonded to the side-chain of D374, the D of the GDAxH motif. The isoalloxazine ring can form π − π interactions with the F354 side chain (backbone omitted for clarity). These residues are highly conserved in the Coq6 family ( S5 Fig ; highlighted in green in Fig 2 ), with the exception of M62 and F203.

    Article Snippet: [ ] This process was facilitated by a reference multiple sequence alignment (MSA) of 119 Coq6p sequence homologs obtained by using the ConSurf server.

    Techniques:

    Sequence alignment of Coq6p with templates used by the different homology modeling approaches. The structural annotations are derived from studies of pHBH, the holotype for this class of enzymes. The templates are referred to by their PDB codes. 2X3N is a putative quinolone monooxygenase from Pseudomonas aeruginosa (1.75 Å resolution); 4N9X, a putative ubiquinone biosynthesis monooxygenase from Erwinia carotovora (2.5 Å resolution); 4K22, a ubiquinone biosynthesis hydroxylase from E . coli (2 Å resolution); 1PBE, the para-hydroxybenzoate hydroxylase from Pseudomonas fluorescens . Secondary structure as calculated by DSSP is indicated by text color: beta strands are blue, alpha helices are red, and turns are in black. Residues missing from crystal structures have background highlighted in red. Residues selected for building the active-site geometry descriptor and scoring function are highlighted in yellow. FAD binding residues are highlighted in green.

    Journal: PLoS Computational Biology

    Article Title: Coenzyme Q Biosynthesis: Evidence for a Substrate Access Channel in the FAD-Dependent Monooxygenase Coq6

    doi: 10.1371/journal.pcbi.1004690

    Figure Lengend Snippet: Sequence alignment of Coq6p with templates used by the different homology modeling approaches. The structural annotations are derived from studies of pHBH, the holotype for this class of enzymes. The templates are referred to by their PDB codes. 2X3N is a putative quinolone monooxygenase from Pseudomonas aeruginosa (1.75 Å resolution); 4N9X, a putative ubiquinone biosynthesis monooxygenase from Erwinia carotovora (2.5 Å resolution); 4K22, a ubiquinone biosynthesis hydroxylase from E . coli (2 Å resolution); 1PBE, the para-hydroxybenzoate hydroxylase from Pseudomonas fluorescens . Secondary structure as calculated by DSSP is indicated by text color: beta strands are blue, alpha helices are red, and turns are in black. Residues missing from crystal structures have background highlighted in red. Residues selected for building the active-site geometry descriptor and scoring function are highlighted in yellow. FAD binding residues are highlighted in green.

    Article Snippet: [ ] This process was facilitated by a reference multiple sequence alignment (MSA) of 119 Coq6p sequence homologs obtained by using the ConSurf server.

    Techniques: Sequencing, Derivative Assay, Binding Assay

    Comparison of pHBH’s active site crystal structure with the same region in the Coq6p model (here, the Coq6p_MODELLER model). The active site interatomic distances used for the scoring function-based substrate docking are shown as blue lines (see Methods ). A): The co-crystal structure of para-hydroxybenzoate in pHBH (1PBE) provides coordinates of a structurally and functionally homologous enzyme-substrate-co-factor complex. In order to capture the active-site geometry compatible with substrate binding, a receptor-based geometric descriptor is derived from the enzyme’s hydrogen bonding partners with the substrate. B): Residues and atoms used in the geometric descriptor derived for pHBH are mapped onto homologous residues and atoms in the Coq6p catalytic site.

    Journal: PLoS Computational Biology

    Article Title: Coenzyme Q Biosynthesis: Evidence for a Substrate Access Channel in the FAD-Dependent Monooxygenase Coq6

    doi: 10.1371/journal.pcbi.1004690

    Figure Lengend Snippet: Comparison of pHBH’s active site crystal structure with the same region in the Coq6p model (here, the Coq6p_MODELLER model). The active site interatomic distances used for the scoring function-based substrate docking are shown as blue lines (see Methods ). A): The co-crystal structure of para-hydroxybenzoate in pHBH (1PBE) provides coordinates of a structurally and functionally homologous enzyme-substrate-co-factor complex. In order to capture the active-site geometry compatible with substrate binding, a receptor-based geometric descriptor is derived from the enzyme’s hydrogen bonding partners with the substrate. B): Residues and atoms used in the geometric descriptor derived for pHBH are mapped onto homologous residues and atoms in the Coq6p catalytic site.

    Article Snippet: [ ] This process was facilitated by a reference multiple sequence alignment (MSA) of 119 Coq6p sequence homologs obtained by using the ConSurf server.

    Techniques: Binding Assay, Derivative Assay

    Recurrent catalytically plausible poses of the 3-hexaprenyl-4-hydroxyphenol model substrate in the re face tunnel 1 of the Coq6p_MODELLER model. The enzyme conformation for the substrate docking calculations was selected with the active site geometric descriptor scoring-function derived from 1PBE[ 40 ] (see Methods ). The FAD co-factor is represented in green stick and the substrate in grey (aromatic head) and blue (isoprenyl chain). Several substrate poses are superimposed within the accessible volume (transparent pink) delimited by the re face substrate access tunne l 1. The substrate’s C1 and C6 hydroxyl groups can form hydrogen bonds (green dashed lines) with the backbone oxygen of P381 and the side chain oxygen of T261. One pose is also illustrated that shows a distance between the substrate’s C5 carbon (the hydroxylation target) and the FAD’s C4X (which bears the reactive peroxo group) of 4.7Å (dashed orange line), similar to the homologous distance observed in the 1PBE structure of 4.3Å.

    Journal: PLoS Computational Biology

    Article Title: Coenzyme Q Biosynthesis: Evidence for a Substrate Access Channel in the FAD-Dependent Monooxygenase Coq6

    doi: 10.1371/journal.pcbi.1004690

    Figure Lengend Snippet: Recurrent catalytically plausible poses of the 3-hexaprenyl-4-hydroxyphenol model substrate in the re face tunnel 1 of the Coq6p_MODELLER model. The enzyme conformation for the substrate docking calculations was selected with the active site geometric descriptor scoring-function derived from 1PBE[ 40 ] (see Methods ). The FAD co-factor is represented in green stick and the substrate in grey (aromatic head) and blue (isoprenyl chain). Several substrate poses are superimposed within the accessible volume (transparent pink) delimited by the re face substrate access tunne l 1. The substrate’s C1 and C6 hydroxyl groups can form hydrogen bonds (green dashed lines) with the backbone oxygen of P381 and the side chain oxygen of T261. One pose is also illustrated that shows a distance between the substrate’s C5 carbon (the hydroxylation target) and the FAD’s C4X (which bears the reactive peroxo group) of 4.7Å (dashed orange line), similar to the homologous distance observed in the 1PBE structure of 4.3Å.

    Article Snippet: [ ] This process was facilitated by a reference multiple sequence alignment (MSA) of 119 Coq6p sequence homologs obtained by using the ConSurf server.

    Techniques: Derivative Assay

    Characterization of the FNDC5 antibody. Western blot of mouse serum (1, 2), murine M. quadriceps femoris (3, 4), bovine plasma (5, 6), bovine M. longissimus (7), and bovine M. semitendinosus (8) with antibody against C-terminus of FNDC5 (left panel). The specific band of ∼25 kDa is marked by a red arrow. The right panel shows a parallel blot where the antibody was blocked with the recombinant antigen peptide prior to incubation. This antibody was raised against aa 149–178 of human FNDC5 corresponding to aa 143–171 in cattle ( Figure 3 , transcript T1–T1).

    Journal: PLoS ONE

    Article Title: Locus Characterization and Gene Expression of Bovine FNDC5: Is the Myokine Irisin Relevant in Cattle?

    doi: 10.1371/journal.pone.0088060

    Figure Lengend Snippet: Characterization of the FNDC5 antibody. Western blot of mouse serum (1, 2), murine M. quadriceps femoris (3, 4), bovine plasma (5, 6), bovine M. longissimus (7), and bovine M. semitendinosus (8) with antibody against C-terminus of FNDC5 (left panel). The specific band of ∼25 kDa is marked by a red arrow. The right panel shows a parallel blot where the antibody was blocked with the recombinant antigen peptide prior to incubation. This antibody was raised against aa 149–178 of human FNDC5 corresponding to aa 143–171 in cattle ( Figure 3 , transcript T1–T1).

    Article Snippet: In a first experiment, we did not detect a specific band neither at 12 kDa (irisin) nor at 25 kDa (FNDC5) in any sample with an antibody directed against a portion of irisin and the C-terminal FNDC5 sequence (Sigma-Aldrich, aa 90–144 see ; data not shown).

    Techniques: Western Blot, Recombinant, Incubation

    Detection of irisin and FNDC5 in bovine and murine skeletal muscle. Western blot of M. longissimus of bulls with high (H) or low (L) muscularity with an antibody against full-length irisin (upper panel). M: murine M. quadriceps femoris. The red arrow marks irisin at ∼12 kDa.The blot was stripped and re-incubated with an antibody against the C-terminus of FNDC5 (lower panel). A single, specific band was detected in bovine and murine muscle. Alpha-tubulin was used for normalization. No differences were found between the groups “low” and “high muscularity” (0.92±0.07 vs. 1.10±0.14; p = 0.27).

    Journal: PLoS ONE

    Article Title: Locus Characterization and Gene Expression of Bovine FNDC5: Is the Myokine Irisin Relevant in Cattle?

    doi: 10.1371/journal.pone.0088060

    Figure Lengend Snippet: Detection of irisin and FNDC5 in bovine and murine skeletal muscle. Western blot of M. longissimus of bulls with high (H) or low (L) muscularity with an antibody against full-length irisin (upper panel). M: murine M. quadriceps femoris. The red arrow marks irisin at ∼12 kDa.The blot was stripped and re-incubated with an antibody against the C-terminus of FNDC5 (lower panel). A single, specific band was detected in bovine and murine muscle. Alpha-tubulin was used for normalization. No differences were found between the groups “low” and “high muscularity” (0.92±0.07 vs. 1.10±0.14; p = 0.27).

    Article Snippet: In a first experiment, we did not detect a specific band neither at 12 kDa (irisin) nor at 25 kDa (FNDC5) in any sample with an antibody directed against a portion of irisin and the C-terminal FNDC5 sequence (Sigma-Aldrich, aa 90–144 see ; data not shown).

    Techniques: Western Blot, Incubation

    Detection of irisin in bovine plasma and murine serum. Western blot of plasma from bulls with high (H) or low (L) muscularity with an antibody against full-length irisin (upper panel). M: murine serum. x: empty lanes. A band specific for irisin was observed only in murine serum and is marked by a red arrow. The blot was stripped and re-incubated with the antibody against the C-terminus of FNDC5 (lower panel). A specific band was detected neither in bovine plasma nor in murine serum.

    Journal: PLoS ONE

    Article Title: Locus Characterization and Gene Expression of Bovine FNDC5: Is the Myokine Irisin Relevant in Cattle?

    doi: 10.1371/journal.pone.0088060

    Figure Lengend Snippet: Detection of irisin in bovine plasma and murine serum. Western blot of plasma from bulls with high (H) or low (L) muscularity with an antibody against full-length irisin (upper panel). M: murine serum. x: empty lanes. A band specific for irisin was observed only in murine serum and is marked by a red arrow. The blot was stripped and re-incubated with the antibody against the C-terminus of FNDC5 (lower panel). A specific band was detected neither in bovine plasma nor in murine serum.

    Article Snippet: In a first experiment, we did not detect a specific band neither at 12 kDa (irisin) nor at 25 kDa (FNDC5) in any sample with an antibody directed against a portion of irisin and the C-terminal FNDC5 sequence (Sigma-Aldrich, aa 90–144 see ; data not shown).

    Techniques: Western Blot, Incubation

    Bovine FNDC5 protein variants and antibody binding. ( A ) Deduced amino acid sequences for different transcripts and transcript fragments identified in this study. Putative signal peptides are underlined and the irisin peptide is marked in bold letters. Amino acids 25-204 in transcript T1–T1 are identical with murine and human amino acid sequences. ( B ) Schematic representation of antibody binding in the bovine FNDC5 protein and irisin peptide, respectively. The antibodies I, II, and III are further described in Materials and Methods. Annotation of functional domains and irisin was borrowed from the murine protein [1] , [3] . SP: signal peptide; H: hydrophobic domain; C: C-terminal domain. Numbers refer to amino acids of bovine full length transcript T1–T1 (see A).

    Journal: PLoS ONE

    Article Title: Locus Characterization and Gene Expression of Bovine FNDC5: Is the Myokine Irisin Relevant in Cattle?

    doi: 10.1371/journal.pone.0088060

    Figure Lengend Snippet: Bovine FNDC5 protein variants and antibody binding. ( A ) Deduced amino acid sequences for different transcripts and transcript fragments identified in this study. Putative signal peptides are underlined and the irisin peptide is marked in bold letters. Amino acids 25-204 in transcript T1–T1 are identical with murine and human amino acid sequences. ( B ) Schematic representation of antibody binding in the bovine FNDC5 protein and irisin peptide, respectively. The antibodies I, II, and III are further described in Materials and Methods. Annotation of functional domains and irisin was borrowed from the murine protein [1] , [3] . SP: signal peptide; H: hydrophobic domain; C: C-terminal domain. Numbers refer to amino acids of bovine full length transcript T1–T1 (see A).

    Article Snippet: In a first experiment, we did not detect a specific band neither at 12 kDa (irisin) nor at 25 kDa (FNDC5) in any sample with an antibody directed against a portion of irisin and the C-terminal FNDC5 sequence (Sigma-Aldrich, aa 90–144 see ; data not shown).

    Techniques: Binding Assay, Functional Assay

    Annotation of the bovine and human FNDC5 genes. ( A ) Bovine FNDC5 locus as annotated in the current build of the bovine genome (UMD 3.1). ( B ) Structure of the human FNDC5 locus. ( C ) Annotated transcripts of the human FNDC5 gene (GenBank accession numbers NM_001171941.1, NM_153756.2, NM_001171940.1). Boxes denote exons with putative coding regions in black. Exon numbers are given below the boxes with lengths in base pairs. Intron lengths are given above the line. Dashed boxes indicate parts of exons which may encode amino acids when the alternative start codon in the human gene is used. The nucleotide numbers of the genomic sequences are given above the arrows.

    Journal: PLoS ONE

    Article Title: Locus Characterization and Gene Expression of Bovine FNDC5: Is the Myokine Irisin Relevant in Cattle?

    doi: 10.1371/journal.pone.0088060

    Figure Lengend Snippet: Annotation of the bovine and human FNDC5 genes. ( A ) Bovine FNDC5 locus as annotated in the current build of the bovine genome (UMD 3.1). ( B ) Structure of the human FNDC5 locus. ( C ) Annotated transcripts of the human FNDC5 gene (GenBank accession numbers NM_001171941.1, NM_153756.2, NM_001171940.1). Boxes denote exons with putative coding regions in black. Exon numbers are given below the boxes with lengths in base pairs. Intron lengths are given above the line. Dashed boxes indicate parts of exons which may encode amino acids when the alternative start codon in the human gene is used. The nucleotide numbers of the genomic sequences are given above the arrows.

    Article Snippet: In a first experiment, we did not detect a specific band neither at 12 kDa (irisin) nor at 25 kDa (FNDC5) in any sample with an antibody directed against a portion of irisin and the C-terminal FNDC5 sequence (Sigma-Aldrich, aa 90–144 see ; data not shown).

    Techniques: Genomic Sequencing

    Cellular localization of bovine FNDC5 in skeletal muscle. Immuno-histochemical detection of FNDC5 in a cross-section of bovine M. semitendinosus (SM). FNDC5 was detected with the antibody against the C-terminus and was located at sarcolemma and in cytosol.

    Journal: PLoS ONE

    Article Title: Locus Characterization and Gene Expression of Bovine FNDC5: Is the Myokine Irisin Relevant in Cattle?

    doi: 10.1371/journal.pone.0088060

    Figure Lengend Snippet: Cellular localization of bovine FNDC5 in skeletal muscle. Immuno-histochemical detection of FNDC5 in a cross-section of bovine M. semitendinosus (SM). FNDC5 was detected with the antibody against the C-terminus and was located at sarcolemma and in cytosol.

    Article Snippet: In a first experiment, we did not detect a specific band neither at 12 kDa (irisin) nor at 25 kDa (FNDC5) in any sample with an antibody directed against a portion of irisin and the C-terminal FNDC5 sequence (Sigma-Aldrich, aa 90–144 see ; data not shown).

    Techniques:

    Characterization of the irisin antibody. Western blot of bovine plasma (1, 2), LM (3), and SM (4) with an antibody against full-length irisin. Recombinant irisin (5) was used as positive control (red arrow; left panel). The right panel shows a parallel blot where the antibody was blocked with recombinant irisin prior to incubation. The antibody was raised against aa 32–143 of human FNDC5 (corresponding to aa 25–136 in cattle; Figure 3 , transcript T1–T1).

    Journal: PLoS ONE

    Article Title: Locus Characterization and Gene Expression of Bovine FNDC5: Is the Myokine Irisin Relevant in Cattle?

    doi: 10.1371/journal.pone.0088060

    Figure Lengend Snippet: Characterization of the irisin antibody. Western blot of bovine plasma (1, 2), LM (3), and SM (4) with an antibody against full-length irisin. Recombinant irisin (5) was used as positive control (red arrow; left panel). The right panel shows a parallel blot where the antibody was blocked with recombinant irisin prior to incubation. The antibody was raised against aa 32–143 of human FNDC5 (corresponding to aa 25–136 in cattle; Figure 3 , transcript T1–T1).

    Article Snippet: In a first experiment, we did not detect a specific band neither at 12 kDa (irisin) nor at 25 kDa (FNDC5) in any sample with an antibody directed against a portion of irisin and the C-terminal FNDC5 sequence (Sigma-Aldrich, aa 90–144 see ; data not shown).

    Techniques: Western Blot, Recombinant, Positive Control, Incubation

    Gene model for bovine FNDC5. ( A ) Genomic organization of bovine FNDC5 on BTA2. Exon 2 (from GenBank accession number EV695596.1, bold line) was anchored to the bovine genome with an overlapping genomic sequence (GenBank accession number AC219791.1, dashed line) and was placed into a gap of the bovine genome. A hitherto unknown exon was named 3c. ( B ) Transcripts identified in bovine skeletal muscle in this study. Boxes denote exons with putative coding regions in black. Grey boxes indicate the proportion of 5′- and 3′-exons which were sequenced in this study. Exon numbers are used like for the human gene and are given below the boxes with lengths in base pairs. Intron lengths are given above the line. Two putative start codons are indicated.

    Journal: PLoS ONE

    Article Title: Locus Characterization and Gene Expression of Bovine FNDC5: Is the Myokine Irisin Relevant in Cattle?

    doi: 10.1371/journal.pone.0088060

    Figure Lengend Snippet: Gene model for bovine FNDC5. ( A ) Genomic organization of bovine FNDC5 on BTA2. Exon 2 (from GenBank accession number EV695596.1, bold line) was anchored to the bovine genome with an overlapping genomic sequence (GenBank accession number AC219791.1, dashed line) and was placed into a gap of the bovine genome. A hitherto unknown exon was named 3c. ( B ) Transcripts identified in bovine skeletal muscle in this study. Boxes denote exons with putative coding regions in black. Grey boxes indicate the proportion of 5′- and 3′-exons which were sequenced in this study. Exon numbers are used like for the human gene and are given below the boxes with lengths in base pairs. Intron lengths are given above the line. Two putative start codons are indicated.

    Article Snippet: In a first experiment, we did not detect a specific band neither at 12 kDa (irisin) nor at 25 kDa (FNDC5) in any sample with an antibody directed against a portion of irisin and the C-terminal FNDC5 sequence (Sigma-Aldrich, aa 90–144 see ; data not shown).

    Techniques: Sequencing