brg1 antibody Abcam Search Results


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  • 93
    Millipore brg1 specific antibodies
    <t>BRG1</t> remodels chromatin at the COUP-TFII promoter and influences accessibility of transcriptional machinery. ( A , B ) C166 endothelial cells were transfected with nonspecific (NS) or BRG1-specific siRNA for 24 hours prior to processing for ChIP assays. (A) ChIP with an antibody against total histone H3 was used to determine nucleosome density at various regions of the COUP-TFII promoter. Nucleosome density was significantly decreased at the -4.7 kb region of the COUP-TFII promoter but was significantly increased at the -1.2 kb and -0.3 kb promoter regions following BRG1 knockdown. (B) ChIP with an antibody against RNA polymerase II (RNAPolII) indicated its ability to bind the -0.3 kb region of the COUP-TFII promoter was significantly decreased following BRG1 knockdown. For A and B, a region upstream of the Fzd5 promoter ( Fzd5 UP ) was used as a negative control region, and the Adamts1 promoter served as a positive control region for BRG1-induced changes in nucleosome density or RNAPolII binding, respectively. Data from three independent experiments were combined and are presented as fold enrichment over the levels of ChIP with the H3 or RNAPolII antibodies in NS siRNA transfected cells±s.e.m. Significant differences were calculated using a two-tailed Student’s t test ( * P
    Brg1 Specific Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Abcam anti brg1 abcam antibody
    GATA1 binding is positively correlated with <t>BRG1</t> binding during differentiation of HSCs. ( A ) UCSC Genome Browser images showing the colocalization of BRG1 with GATA1 in CD36 + cells. The four DNase I hypersensitive sites (HS) bound by GATA1 in the LCR
    Anti Brg1 Abcam Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti brg1 epncir111a abcam
    ARID1A loss impairs enhancer-mediated gene regulation in the colonic epithelium a) Observed vs. expected TF motif instances at TSS-distal H3K27ac regions (enhancers) with reduced H3K27ac signal based on enrichment regions with stable H3K27ac signal. Motifs highly similar to AP1 and CTCF motifs are highlighted; b) Correlation between H3K27ac signal change ( Arid1a −/− / WT) and WT ChIP-Seq signal levels of different factors profiled in this work and by the ENCODE Project. c) ChIP-Seq profiles for <t>SMARCA4,</t> SMARCC1, JUND, FOSL1, and CTCF in WT HCT116 cells centered around TSS-distal H3K27ac regions (enhancers) that remain stable, show lost/weakened H3K27ac, or show gained/strengthened H3K27ac in Arid1a −/− cells relative to WT; d) H3K27ac levels at TSS-proximal (promoter) and TSS-distal (enhancer) enrichment regions for colon epithelium from wildtype (WT) and Villin-Cre ER-T2 Arid1a fl/fl ( Arid1a −/− ) mice (Note: numbers in the three corners denote numbers of activated ( > 2×), inactivated (
    Anti Brg1 Epncir111a Abcam, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam anti smarca4 brg1 n terminus
    ARID1A loss impairs enhancer-mediated gene regulation in the colonic epithelium a) Observed vs. expected TF motif instances at TSS-distal H3K27ac regions (enhancers) with reduced H3K27ac signal based on enrichment regions with stable H3K27ac signal. Motifs highly similar to AP1 and CTCF motifs are highlighted; b) Correlation between H3K27ac signal change ( Arid1a −/− / WT) and WT ChIP-Seq signal levels of different factors profiled in this work and by the ENCODE Project. c) ChIP-Seq profiles for <t>SMARCA4,</t> SMARCC1, JUND, FOSL1, and CTCF in WT HCT116 cells centered around TSS-distal H3K27ac regions (enhancers) that remain stable, show lost/weakened H3K27ac, or show gained/strengthened H3K27ac in Arid1a −/− cells relative to WT; d) H3K27ac levels at TSS-proximal (promoter) and TSS-distal (enhancer) enrichment regions for colon epithelium from wildtype (WT) and Villin-Cre ER-T2 Arid1a fl/fl ( Arid1a −/− ) mice (Note: numbers in the three corners denote numbers of activated ( > 2×), inactivated (
    Anti Smarca4 Brg1 N Terminus, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology anti smarca4 brg1
    ARID1A loss impairs enhancer-mediated gene regulation in the colonic epithelium a) Observed vs. expected TF motif instances at TSS-distal H3K27ac regions (enhancers) with reduced H3K27ac signal based on enrichment regions with stable H3K27ac signal. Motifs highly similar to AP1 and CTCF motifs are highlighted; b) Correlation between H3K27ac signal change ( Arid1a −/− / WT) and WT ChIP-Seq signal levels of different factors profiled in this work and by the ENCODE Project. c) ChIP-Seq profiles for <t>SMARCA4,</t> SMARCC1, JUND, FOSL1, and CTCF in WT HCT116 cells centered around TSS-distal H3K27ac regions (enhancers) that remain stable, show lost/weakened H3K27ac, or show gained/strengthened H3K27ac in Arid1a −/− cells relative to WT; d) H3K27ac levels at TSS-proximal (promoter) and TSS-distal (enhancer) enrichment regions for colon epithelium from wildtype (WT) and Villin-Cre ER-T2 Arid1a fl/fl ( Arid1a −/− ) mice (Note: numbers in the three corners denote numbers of activated ( > 2×), inactivated (
    Anti Smarca4 Brg1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology brg1
    Foxa2/H2A.Z-driven nucleosome depletion complexes during ES cell differentiation. ( A–C ) <t>Smarca4</t> <t>(Brg1),</t> Kat5 (Tip60), and Nap1l1, are enriched at nucleosome depletion regions. The tag density was normalized to 10 million sequencing tags for each
    Brg1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1513 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam brg1 biotin
    Foxa2/H2A.Z-driven nucleosome depletion complexes during ES cell differentiation. ( A–C ) <t>Smarca4</t> <t>(Brg1),</t> Kat5 (Tip60), and Nap1l1, are enriched at nucleosome depletion regions. The tag density was normalized to 10 million sequencing tags for each
    Brg1 Biotin, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    BRG1 remodels chromatin at the COUP-TFII promoter and influences accessibility of transcriptional machinery. ( A , B ) C166 endothelial cells were transfected with nonspecific (NS) or BRG1-specific siRNA for 24 hours prior to processing for ChIP assays. (A) ChIP with an antibody against total histone H3 was used to determine nucleosome density at various regions of the COUP-TFII promoter. Nucleosome density was significantly decreased at the -4.7 kb region of the COUP-TFII promoter but was significantly increased at the -1.2 kb and -0.3 kb promoter regions following BRG1 knockdown. (B) ChIP with an antibody against RNA polymerase II (RNAPolII) indicated its ability to bind the -0.3 kb region of the COUP-TFII promoter was significantly decreased following BRG1 knockdown. For A and B, a region upstream of the Fzd5 promoter ( Fzd5 UP ) was used as a negative control region, and the Adamts1 promoter served as a positive control region for BRG1-induced changes in nucleosome density or RNAPolII binding, respectively. Data from three independent experiments were combined and are presented as fold enrichment over the levels of ChIP with the H3 or RNAPolII antibodies in NS siRNA transfected cells±s.e.m. Significant differences were calculated using a two-tailed Student’s t test ( * P

    Journal: Development (Cambridge, England)

    Article Title: BRG1 promotes COUP-TFII expression and venous specification during embryonic vascular development

    doi: 10.1242/dev.087379

    Figure Lengend Snippet: BRG1 remodels chromatin at the COUP-TFII promoter and influences accessibility of transcriptional machinery. ( A , B ) C166 endothelial cells were transfected with nonspecific (NS) or BRG1-specific siRNA for 24 hours prior to processing for ChIP assays. (A) ChIP with an antibody against total histone H3 was used to determine nucleosome density at various regions of the COUP-TFII promoter. Nucleosome density was significantly decreased at the -4.7 kb region of the COUP-TFII promoter but was significantly increased at the -1.2 kb and -0.3 kb promoter regions following BRG1 knockdown. (B) ChIP with an antibody against RNA polymerase II (RNAPolII) indicated its ability to bind the -0.3 kb region of the COUP-TFII promoter was significantly decreased following BRG1 knockdown. For A and B, a region upstream of the Fzd5 promoter ( Fzd5 UP ) was used as a negative control region, and the Adamts1 promoter served as a positive control region for BRG1-induced changes in nucleosome density or RNAPolII binding, respectively. Data from three independent experiments were combined and are presented as fold enrichment over the levels of ChIP with the H3 or RNAPolII antibodies in NS siRNA transfected cells±s.e.m. Significant differences were calculated using a two-tailed Student’s t test ( * P

    Article Snippet: A mixture of BRG1-specific antibodies (Millipore, 07-478; Abcam, ab4081) was used to immunoprecipitate protein-DNA complexes.

    Techniques: Transfection, Chromatin Immunoprecipitation, Negative Control, Positive Control, Binding Assay, Two Tailed Test

    BRG1 binds to the COUP-TFII promoter in endothelial cells. ( A ) Alignment of the murine COUP-TFII ). Peak heights indicate degree of sequence homology; pink bars above peaks denote evolutionarily conserved regions; yellow represents the COUP-TFII 5′ untranslated region; blue indicates COUP-TFII exon 1. Boxed regions were selected for further analysis of BRG1 binding. Numbers above boxed regions denote approximate distances upstream of the COUP-TFII transcription start site (TSS). ( B ) Chromatin immunoprecipitation (ChIP) assays were performed on C166 endothelial cells using antibodies against BRG1 or isotype-matched non-specific IgG as a negative control. DNA was isolated and amplified by qPCR to determine whether BRG1 bound to various COUP-TFII promoter regions. Significant BRG1 binding was detected at the -0.3 kb, the -1.2 kb and the -4.7 kb promoter regions. A region upstream of the Fzd5 promoter ( Fzd5 UP ) was used as a negative control BRG1-binding region, and the Adamts1 ). Data from four independent experiments were combined and are presented as fold enrichment over the level of ChIP with negative control IgG antibodies±s.e.m. Significant differences were calculated using a two-tailed Student’s t -test ( * P

    Journal: Development (Cambridge, England)

    Article Title: BRG1 promotes COUP-TFII expression and venous specification during embryonic vascular development

    doi: 10.1242/dev.087379

    Figure Lengend Snippet: BRG1 binds to the COUP-TFII promoter in endothelial cells. ( A ) Alignment of the murine COUP-TFII ). Peak heights indicate degree of sequence homology; pink bars above peaks denote evolutionarily conserved regions; yellow represents the COUP-TFII 5′ untranslated region; blue indicates COUP-TFII exon 1. Boxed regions were selected for further analysis of BRG1 binding. Numbers above boxed regions denote approximate distances upstream of the COUP-TFII transcription start site (TSS). ( B ) Chromatin immunoprecipitation (ChIP) assays were performed on C166 endothelial cells using antibodies against BRG1 or isotype-matched non-specific IgG as a negative control. DNA was isolated and amplified by qPCR to determine whether BRG1 bound to various COUP-TFII promoter regions. Significant BRG1 binding was detected at the -0.3 kb, the -1.2 kb and the -4.7 kb promoter regions. A region upstream of the Fzd5 promoter ( Fzd5 UP ) was used as a negative control BRG1-binding region, and the Adamts1 ). Data from four independent experiments were combined and are presented as fold enrichment over the level of ChIP with negative control IgG antibodies±s.e.m. Significant differences were calculated using a two-tailed Student’s t -test ( * P

    Article Snippet: A mixture of BRG1-specific antibodies (Millipore, 07-478; Abcam, ab4081) was used to immunoprecipitate protein-DNA complexes.

    Techniques: Sequencing, Binding Assay, Chromatin Immunoprecipitation, Negative Control, Isolation, Amplification, Real-time Polymerase Chain Reaction, Two Tailed Test

    Brg1 fl/fl :Tie2-Cre + veins express arterial markers. ( A , B ) Control and Brg1 fl/fl :Tie2-Cre + embryos were crossed onto an Efnb2 LacZ arterial reporter line and stained with X-gal solution to reveal sites of Efnb2 ( LacZ ) expression (blue). Flat-mounted E9.5 yolk sacs displayed aberrant Efnb2 expression in Brg1 fl/fl ;Efnb2 LacZ :Tie2-Cre + veins (B) compared with control veins (A). ( C-F ) E9.75 littermate control and Brg1 fl/fl :Tie2-Cre + embryos were cross-sectioned, and sections containing a dorsal aorta (D.A.) and cardinal vein (C.V.) were immunostained for arterial markers. (C,D) Sections were stained for the endothelial cell marker PECAM1 (red), the arterial marker NRP1 (green) and the nuclear marker Hoechst (blue). NRP1 was aberrantly upregulated on endothelial cells within Brg1 fl/fl :Tie2-Cre + cardinal veins (arrowheads in D). (E,F) Sections were stained for PECAM1 (red), DLL4 (green) and Hoechst (blue). Arterial DLL4 was likewise upregulated on Brg1 fl/fl :Tie2-Cre + cardinal veins (see arrowheads in F). Scale bars: 100 μm.

    Journal: Development (Cambridge, England)

    Article Title: BRG1 promotes COUP-TFII expression and venous specification during embryonic vascular development

    doi: 10.1242/dev.087379

    Figure Lengend Snippet: Brg1 fl/fl :Tie2-Cre + veins express arterial markers. ( A , B ) Control and Brg1 fl/fl :Tie2-Cre + embryos were crossed onto an Efnb2 LacZ arterial reporter line and stained with X-gal solution to reveal sites of Efnb2 ( LacZ ) expression (blue). Flat-mounted E9.5 yolk sacs displayed aberrant Efnb2 expression in Brg1 fl/fl ;Efnb2 LacZ :Tie2-Cre + veins (B) compared with control veins (A). ( C-F ) E9.75 littermate control and Brg1 fl/fl :Tie2-Cre + embryos were cross-sectioned, and sections containing a dorsal aorta (D.A.) and cardinal vein (C.V.) were immunostained for arterial markers. (C,D) Sections were stained for the endothelial cell marker PECAM1 (red), the arterial marker NRP1 (green) and the nuclear marker Hoechst (blue). NRP1 was aberrantly upregulated on endothelial cells within Brg1 fl/fl :Tie2-Cre + cardinal veins (arrowheads in D). (E,F) Sections were stained for PECAM1 (red), DLL4 (green) and Hoechst (blue). Arterial DLL4 was likewise upregulated on Brg1 fl/fl :Tie2-Cre + cardinal veins (see arrowheads in F). Scale bars: 100 μm.

    Article Snippet: A mixture of BRG1-specific antibodies (Millipore, 07-478; Abcam, ab4081) was used to immunoprecipitate protein-DNA complexes.

    Techniques: Staining, Expressing, Marker

    Brg1 fl/fl :Tie2-Cre + yolk sac veins are morphologically abnormal. ( A-D ) Anti-PECAM1 staining on flat-mounted E9.5 yolk sacs revealed Brg1 fl/fl :Tie2-Cre + veins were more abnormal than arteries. Whereas the arterial sides of control and Brg1 fl/fl :Tie2-Cre + yolk sacs contained branching networks of vessels, the venous side of Brg1 fl/fl :Tie2-Cre + yolk sacs failed to undergo normal branching and development. (C,D) Magnified views of the boxed regions in A,B, respectively, including vitelline veins (V.V.). Arrows in D indicate Brg1 fl/fl :Tie2-Cre + veins that failed to interconnect or underwent aberrant regression. Asterisks indicate round, non-vascular spaces that are characteristic of failed vascular plexus remodeling. Scale bars: 500 μm.

    Journal: Development (Cambridge, England)

    Article Title: BRG1 promotes COUP-TFII expression and venous specification during embryonic vascular development

    doi: 10.1242/dev.087379

    Figure Lengend Snippet: Brg1 fl/fl :Tie2-Cre + yolk sac veins are morphologically abnormal. ( A-D ) Anti-PECAM1 staining on flat-mounted E9.5 yolk sacs revealed Brg1 fl/fl :Tie2-Cre + veins were more abnormal than arteries. Whereas the arterial sides of control and Brg1 fl/fl :Tie2-Cre + yolk sacs contained branching networks of vessels, the venous side of Brg1 fl/fl :Tie2-Cre + yolk sacs failed to undergo normal branching and development. (C,D) Magnified views of the boxed regions in A,B, respectively, including vitelline veins (V.V.). Arrows in D indicate Brg1 fl/fl :Tie2-Cre + veins that failed to interconnect or underwent aberrant regression. Asterisks indicate round, non-vascular spaces that are characteristic of failed vascular plexus remodeling. Scale bars: 500 μm.

    Article Snippet: A mixture of BRG1-specific antibodies (Millipore, 07-478; Abcam, ab4081) was used to immunoprecipitate protein-DNA complexes.

    Techniques: Staining

    Brg1 fl/fl :Tie2-Cre + veins aberrantly recruit smooth muscle cells. Tissues from E10.5 littermate control and Brg1 fl/fl :Tie2-Cre + mutants were sectioned and immunostained for the endothelial cell marker PECAM1 (green), the smooth muscle cell marker α-smooth muscle actin (αSMA) (red) and the nuclear marker Hoechst (blue). ( A , B ) Extra-embryonic umbilical vessels, including umbilical veins (U.V.) and umbilical arteries (U.A.), were sectioned and stained. In control vessels, αSMA-positive cells predominantly accumulated around umbilical arteries (A). However, αSMA-positive cells accumulated around both umbilical arteries and veins in Brg1 fl/fl :Tie2-Cre + mutants (B). ( C , D ) Sections of embryos containing a dorsal aorta (D.A.) and cardinal vein (C.V.) were immunostained. αSMA-positive cells were detected around the Brg1 fl/fl :Tie2-Cre + C.V. (arrowheads in magnified inset of D) but not around the control C.V. (C). Scale bars: 100 μm.

    Journal: Development (Cambridge, England)

    Article Title: BRG1 promotes COUP-TFII expression and venous specification during embryonic vascular development

    doi: 10.1242/dev.087379

    Figure Lengend Snippet: Brg1 fl/fl :Tie2-Cre + veins aberrantly recruit smooth muscle cells. Tissues from E10.5 littermate control and Brg1 fl/fl :Tie2-Cre + mutants were sectioned and immunostained for the endothelial cell marker PECAM1 (green), the smooth muscle cell marker α-smooth muscle actin (αSMA) (red) and the nuclear marker Hoechst (blue). ( A , B ) Extra-embryonic umbilical vessels, including umbilical veins (U.V.) and umbilical arteries (U.A.), were sectioned and stained. In control vessels, αSMA-positive cells predominantly accumulated around umbilical arteries (A). However, αSMA-positive cells accumulated around both umbilical arteries and veins in Brg1 fl/fl :Tie2-Cre + mutants (B). ( C , D ) Sections of embryos containing a dorsal aorta (D.A.) and cardinal vein (C.V.) were immunostained. αSMA-positive cells were detected around the Brg1 fl/fl :Tie2-Cre + C.V. (arrowheads in magnified inset of D) but not around the control C.V. (C). Scale bars: 100 μm.

    Article Snippet: A mixture of BRG1-specific antibodies (Millipore, 07-478; Abcam, ab4081) was used to immunoprecipitate protein-DNA complexes.

    Techniques: Marker, Staining

    COUP-TFII expression is downregulated in Brg1 -deficient endothelial cells. (A-D) Cryosections of littermate control and Brg1 fl/fl :Tie2-Cre + tissues were immunostained for the endothelial cell marker PECAM1 (red), COUP-TFII (green) and the nuclear marker Hoechst (blue). ( A , B ) Cross-sectioned E10.5 umbilical vessels were immunostained, and although COUP-TFII was expressed in umbilical vein (U.V.) endothelial cells in the control section (A), it was significantly diminished in Brg1 fl/fl :Tie2-Cre + venous endothelial cells (B). U.A., umbilical artery.( C , D ) E9.75 embryos were cross-sectioned and immunostained. COUP-TFII was expressed in endothelial cells of the cardinal vein (C.V.) in the control section (C) but was downregulated in Brg1 fl/fl :Tie2-Cre + C.V. endothelial cells (D). D.A., dorsal aorta. For A-D, insets show magnified views of the boxed regions and arrowheads indicate individual endothelial cells. Scale bars: 100 μm. ( E , F ) Primary endothelial cells (ECs) were isolated from E10.5 control and Brg1 fl/fl :Tie2-Cre + tissues, RNA was purified and cDNA was synthesized. Samples from individual littermate control and Brg1 fl/fl :Tie2-Cre + yolk sacs (E) or embryos (F) were processed for qPCR analysis of Brg1 and COUP-TFII expression. Data from three independent experiments were combined and are presented as relative fold change over the expression levels in control cells±s.em. Significant differences were calculated using a two-tailed Student’s t -test ( * P

    Journal: Development (Cambridge, England)

    Article Title: BRG1 promotes COUP-TFII expression and venous specification during embryonic vascular development

    doi: 10.1242/dev.087379

    Figure Lengend Snippet: COUP-TFII expression is downregulated in Brg1 -deficient endothelial cells. (A-D) Cryosections of littermate control and Brg1 fl/fl :Tie2-Cre + tissues were immunostained for the endothelial cell marker PECAM1 (red), COUP-TFII (green) and the nuclear marker Hoechst (blue). ( A , B ) Cross-sectioned E10.5 umbilical vessels were immunostained, and although COUP-TFII was expressed in umbilical vein (U.V.) endothelial cells in the control section (A), it was significantly diminished in Brg1 fl/fl :Tie2-Cre + venous endothelial cells (B). U.A., umbilical artery.( C , D ) E9.75 embryos were cross-sectioned and immunostained. COUP-TFII was expressed in endothelial cells of the cardinal vein (C.V.) in the control section (C) but was downregulated in Brg1 fl/fl :Tie2-Cre + C.V. endothelial cells (D). D.A., dorsal aorta. For A-D, insets show magnified views of the boxed regions and arrowheads indicate individual endothelial cells. Scale bars: 100 μm. ( E , F ) Primary endothelial cells (ECs) were isolated from E10.5 control and Brg1 fl/fl :Tie2-Cre + tissues, RNA was purified and cDNA was synthesized. Samples from individual littermate control and Brg1 fl/fl :Tie2-Cre + yolk sacs (E) or embryos (F) were processed for qPCR analysis of Brg1 and COUP-TFII expression. Data from three independent experiments were combined and are presented as relative fold change over the expression levels in control cells±s.em. Significant differences were calculated using a two-tailed Student’s t -test ( * P

    Article Snippet: A mixture of BRG1-specific antibodies (Millipore, 07-478; Abcam, ab4081) was used to immunoprecipitate protein-DNA complexes.

    Techniques: Expressing, Marker, Isolation, Purification, Synthesized, Real-time Polymerase Chain Reaction, Two Tailed Test

    BRG1 impacts expression of genes downstream of COUP-TFII signaling. ( A ) Primary endothelial cells (ECs) were isolated from E10.5 control and Brg1 fl/fl :Tie2-Cre + embryos, RNA was purified, and cDNA was synthesized. Expression levels of Brg1 , the arterial markers (red) Nrp1, Hey1, Dll4, Hey2 and Foxc1 , and the venous markers (blue) COUP-TFII, Ephb4 and Nrp2 were measured by qPCR. Data from three independent experiments were combined and are presented as relative fold change over the normalized expression level of each gene in control cells (dotted line) ±s.e.m. Significant differences were calculated using a two-tailed Student’s t -test ( * P

    Journal: Development (Cambridge, England)

    Article Title: BRG1 promotes COUP-TFII expression and venous specification during embryonic vascular development

    doi: 10.1242/dev.087379

    Figure Lengend Snippet: BRG1 impacts expression of genes downstream of COUP-TFII signaling. ( A ) Primary endothelial cells (ECs) were isolated from E10.5 control and Brg1 fl/fl :Tie2-Cre + embryos, RNA was purified, and cDNA was synthesized. Expression levels of Brg1 , the arterial markers (red) Nrp1, Hey1, Dll4, Hey2 and Foxc1 , and the venous markers (blue) COUP-TFII, Ephb4 and Nrp2 were measured by qPCR. Data from three independent experiments were combined and are presented as relative fold change over the normalized expression level of each gene in control cells (dotted line) ±s.e.m. Significant differences were calculated using a two-tailed Student’s t -test ( * P

    Article Snippet: A mixture of BRG1-specific antibodies (Millipore, 07-478; Abcam, ab4081) was used to immunoprecipitate protein-DNA complexes.

    Techniques: Expressing, Isolation, Purification, Synthesized, Real-time Polymerase Chain Reaction, Two Tailed Test

    GATA1 binding is positively correlated with BRG1 binding during differentiation of HSCs. ( A ) UCSC Genome Browser images showing the colocalization of BRG1 with GATA1 in CD36 + cells. The four DNase I hypersensitive sites (HS) bound by GATA1 in the LCR

    Journal: Genome Research

    Article Title: Regulation of nucleosome landscape and transcription factor targeting at tissue-specific enhancers by BRG1

    doi: 10.1101/gr.121145.111

    Figure Lengend Snippet: GATA1 binding is positively correlated with BRG1 binding during differentiation of HSCs. ( A ) UCSC Genome Browser images showing the colocalization of BRG1 with GATA1 in CD36 + cells. The four DNase I hypersensitive sites (HS) bound by GATA1 in the LCR

    Article Snippet: GFP-positive cells were sorted and further cultured for 8 d before being processed for ChIP-seq analysis using antibodies against BRG1 , H3K4me1 (abcam ab8898), GATA1 (abcam ab11852), TAL1 (Santa Cruz Biotechnology, sc-12984), and CTCF (Upstate, 07-729).

    Techniques: Binding Assay

    BRG1-induced nucleosome shifting facilitates binding of TAL1. ( A ) Venn diagram comparison of TAL1 binding sites in HSCs and CD36 + cells. ( B ) Box-plots for the normalized TAL1 tag density for the three groups of TAL1 sites as specified in A in three cell

    Journal: Genome Research

    Article Title: Regulation of nucleosome landscape and transcription factor targeting at tissue-specific enhancers by BRG1

    doi: 10.1101/gr.121145.111

    Figure Lengend Snippet: BRG1-induced nucleosome shifting facilitates binding of TAL1. ( A ) Venn diagram comparison of TAL1 binding sites in HSCs and CD36 + cells. ( B ) Box-plots for the normalized TAL1 tag density for the three groups of TAL1 sites as specified in A in three cell

    Article Snippet: GFP-positive cells were sorted and further cultured for 8 d before being processed for ChIP-seq analysis using antibodies against BRG1 , H3K4me1 (abcam ab8898), GATA1 (abcam ab11852), TAL1 (Santa Cruz Biotechnology, sc-12984), and CTCF (Upstate, 07-729).

    Techniques: Binding Assay

    BRG1 mediates nucleosome shifting surrounding the GATA1 sites. ( A ) Nucleosome profiles in HSCs surrounding the distal GATA1 binding sites that are identified in CD36 + cells. The sites are grouped into four quartiles according to the level of BRG1 binding

    Journal: Genome Research

    Article Title: Regulation of nucleosome landscape and transcription factor targeting at tissue-specific enhancers by BRG1

    doi: 10.1101/gr.121145.111

    Figure Lengend Snippet: BRG1 mediates nucleosome shifting surrounding the GATA1 sites. ( A ) Nucleosome profiles in HSCs surrounding the distal GATA1 binding sites that are identified in CD36 + cells. The sites are grouped into four quartiles according to the level of BRG1 binding

    Article Snippet: GFP-positive cells were sorted and further cultured for 8 d before being processed for ChIP-seq analysis using antibodies against BRG1 , H3K4me1 (abcam ab8898), GATA1 (abcam ab11852), TAL1 (Santa Cruz Biotechnology, sc-12984), and CTCF (Upstate, 07-729).

    Techniques: Binding Assay

    BRG1 knockdown decreases binding of TAL1 but not GATA1. ( A ) UCSC Genome Browser images of GATA1 binding in the BRG1 knockdown and control cells. Genomic regions with significantly increased and decreased levels of GATA1 binding ( P -value

    Journal: Genome Research

    Article Title: Regulation of nucleosome landscape and transcription factor targeting at tissue-specific enhancers by BRG1

    doi: 10.1101/gr.121145.111

    Figure Lengend Snippet: BRG1 knockdown decreases binding of TAL1 but not GATA1. ( A ) UCSC Genome Browser images of GATA1 binding in the BRG1 knockdown and control cells. Genomic regions with significantly increased and decreased levels of GATA1 binding ( P -value

    Article Snippet: GFP-positive cells were sorted and further cultured for 8 d before being processed for ChIP-seq analysis using antibodies against BRG1 , H3K4me1 (abcam ab8898), GATA1 (abcam ab11852), TAL1 (Santa Cruz Biotechnology, sc-12984), and CTCF (Upstate, 07-729).

    Techniques: Binding Assay

    BRG1 plays a tumor suppressor role. (A) Colony formation of 5637-NC and 5637-iBRG1 cells. The cells were cultured for 14 days. Colonies were stained using crystal violet. (B) Colony formation of 5637-pcDNA3.1 and 5637-BRG1 cells. The cells were cultured for 14 days. Colonies were stained using crystal violet. (C) Growth curves of 5637 cells after transfection with BRG1 RNAi. Cellular proliferation was measured using MTT assay at 24, 48, 72, 96 and 120 h. The differences in data of 72, 96 and 120 h were significant, * P

    Journal: Oncology Reports

    Article Title: Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells

    doi: 10.3892/or.2014.3309

    Figure Lengend Snippet: BRG1 plays a tumor suppressor role. (A) Colony formation of 5637-NC and 5637-iBRG1 cells. The cells were cultured for 14 days. Colonies were stained using crystal violet. (B) Colony formation of 5637-pcDNA3.1 and 5637-BRG1 cells. The cells were cultured for 14 days. Colonies were stained using crystal violet. (C) Growth curves of 5637 cells after transfection with BRG1 RNAi. Cellular proliferation was measured using MTT assay at 24, 48, 72, 96 and 120 h. The differences in data of 72, 96 and 120 h were significant, * P

    Article Snippet: ChIP was performed overnight at 4°C using the BRG1 antibody (ab4081; Abcam) or an isotype control IgG.

    Techniques: Cell Culture, Staining, Transfection, MTT Assay

    UCA1 blocks recruitment of BRG1 to chromatin. (A) UCA1 does not affect the ATPase activity of BRG1. The kinetics of BRG1-induced ATP hydrolysis were analyzed in the presence or absence of UCA1. (B) ChIP analysis of BRG1 binding to the p21 promoter in 5637-iUCA1. 5637-NC cells were used as the control. Genomic DNA was fixed and immunoprecipitated using anti-BRG1 antibody, with IgG as a negative control. Real-time PCR was performed using a primer set specific to the BRG1-binding site of p21 promoter. Data were normalized to input and are expressed as the means ± SD of three independent experiments. * P

    Journal: Oncology Reports

    Article Title: Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells

    doi: 10.3892/or.2014.3309

    Figure Lengend Snippet: UCA1 blocks recruitment of BRG1 to chromatin. (A) UCA1 does not affect the ATPase activity of BRG1. The kinetics of BRG1-induced ATP hydrolysis were analyzed in the presence or absence of UCA1. (B) ChIP analysis of BRG1 binding to the p21 promoter in 5637-iUCA1. 5637-NC cells were used as the control. Genomic DNA was fixed and immunoprecipitated using anti-BRG1 antibody, with IgG as a negative control. Real-time PCR was performed using a primer set specific to the BRG1-binding site of p21 promoter. Data were normalized to input and are expressed as the means ± SD of three independent experiments. * P

    Article Snippet: ChIP was performed overnight at 4°C using the BRG1 antibody (ab4081; Abcam) or an isotype control IgG.

    Techniques: Activity Assay, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction

    UCA1 binds to BRG1 in vitro and in vivo . (A) Electrophoresis of proteins bound to biotin-labeled sense and antisense UCA1 in NuPAGE 4–12% Bis-Tris gel. The biotin-labeled sense and antisense UCA1 were transcribed in vitro and incubated with HeLa cell lysate. The arrow shows the protein band for BRG1. (B) Western blot analysis of biotin-labeled sense and antisense UCA1-bound proteins using antibody against BRG1. (C) UCA1 binding BRG1 in 5637 cells was detected by RNA-binding protein immunoprecipitation assay. Antibody against BRG1 and mouse IgG (as a negative control) were used to pull-down RNAs in 5637 cells. The level of UCA1 was determined using real-time PCR. Data were normalized to input and are expressed as the means ± SD of three independent experiments * P

    Journal: Oncology Reports

    Article Title: Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells

    doi: 10.3892/or.2014.3309

    Figure Lengend Snippet: UCA1 binds to BRG1 in vitro and in vivo . (A) Electrophoresis of proteins bound to biotin-labeled sense and antisense UCA1 in NuPAGE 4–12% Bis-Tris gel. The biotin-labeled sense and antisense UCA1 were transcribed in vitro and incubated with HeLa cell lysate. The arrow shows the protein band for BRG1. (B) Western blot analysis of biotin-labeled sense and antisense UCA1-bound proteins using antibody against BRG1. (C) UCA1 binding BRG1 in 5637 cells was detected by RNA-binding protein immunoprecipitation assay. Antibody against BRG1 and mouse IgG (as a negative control) were used to pull-down RNAs in 5637 cells. The level of UCA1 was determined using real-time PCR. Data were normalized to input and are expressed as the means ± SD of three independent experiments * P

    Article Snippet: ChIP was performed overnight at 4°C using the BRG1 antibody (ab4081; Abcam) or an isotype control IgG.

    Techniques: In Vitro, In Vivo, Electrophoresis, Labeling, Incubation, Western Blot, Binding Assay, RNA Binding Assay, Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction

    UCA1 antagonizes the tumor suppressing function of BRG1. (A) (Left) Colony formation of EJ control, EJ-BRG1 (overexpressed BRG1) and EJ-BRG1-UCA1 (overexpressed BRG1 and UCA1) cells. The cells were cultured with G418 for 10 days. Colonies were stained using crystal violet. (Right) Colony formation of 5637 control, 5637-iUCA1 (UCA1 knocked down) and 5637-iUCA1-iBRG1 (UCA1 and BRG1 both knocked down) cells. The cells were cultured with G418 and puromycin for 10 days. Colonies were stained using crystal violet. (B) (Left) Growth curves of EJ control, EJ-BRG1 and EJ-BRG1-UCA1 cells. Cellular proliferation was measured using MTT assays at 24, 48, 72, 96 and 120 h. The differences between EJ-BRG1 and EJ-BRG1-UCA1 were significant. * P

    Journal: Oncology Reports

    Article Title: Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells

    doi: 10.3892/or.2014.3309

    Figure Lengend Snippet: UCA1 antagonizes the tumor suppressing function of BRG1. (A) (Left) Colony formation of EJ control, EJ-BRG1 (overexpressed BRG1) and EJ-BRG1-UCA1 (overexpressed BRG1 and UCA1) cells. The cells were cultured with G418 for 10 days. Colonies were stained using crystal violet. (Right) Colony formation of 5637 control, 5637-iUCA1 (UCA1 knocked down) and 5637-iUCA1-iBRG1 (UCA1 and BRG1 both knocked down) cells. The cells were cultured with G418 and puromycin for 10 days. Colonies were stained using crystal violet. (B) (Left) Growth curves of EJ control, EJ-BRG1 and EJ-BRG1-UCA1 cells. Cellular proliferation was measured using MTT assays at 24, 48, 72, 96 and 120 h. The differences between EJ-BRG1 and EJ-BRG1-UCA1 were significant. * P

    Article Snippet: ChIP was performed overnight at 4°C using the BRG1 antibody (ab4081; Abcam) or an isotype control IgG.

    Techniques: Cell Culture, Staining, MTT Assay

    UCA1 expression correlates positively with BRG1 expression in bladder cancer tissue samples. (A) Real-time PCR was used to analyze UCA1 and BRG1 mRNA levels in the same samples. S, sample. (B) The status of UCA1 and BRG1 expression in bladder cancer specimens, > 2-fold was identified as high expression. The 2×2 correlation table and Fisher’s exact test were used. The two-sided value P=0.009 was considered significant. UCA1, urothelial carcinoma associated 1.

    Journal: Oncology Reports

    Article Title: Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells

    doi: 10.3892/or.2014.3309

    Figure Lengend Snippet: UCA1 expression correlates positively with BRG1 expression in bladder cancer tissue samples. (A) Real-time PCR was used to analyze UCA1 and BRG1 mRNA levels in the same samples. S, sample. (B) The status of UCA1 and BRG1 expression in bladder cancer specimens, > 2-fold was identified as high expression. The 2×2 correlation table and Fisher’s exact test were used. The two-sided value P=0.009 was considered significant. UCA1, urothelial carcinoma associated 1.

    Article Snippet: ChIP was performed overnight at 4°C using the BRG1 antibody (ab4081; Abcam) or an isotype control IgG.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    BRG1 recruited by PAR regulates chromatin relaxation to facilitate HR. ( A ) Co-IP and WB analysis of the interaction between BRG1 and PAR in response to IR. ( B ) ChIP analysis of BRG1 enrichment at DNA damage sites induced by I-SceI digestion in the presence or absence of the PARP1 inhibitor, olaparib. ( C ) Schematic representation of the BRG1 domain structure, and the truncated constructs used in this study. ( D ) Co-IP and WB analysis of the interaction between PAR and the three domains of BRG1 in the presence or absence of DNA DSBs induced by X-Ray. ( E ) Analysis of recruitment of three BRG1 domains to DSBs using ChIP assay. Different amounts of GFP tagged full length BRG1 and three BRG1 domains were transfected into NHEJ-I9A cells to ensure equal expression of them. Then ChIP was carried out by using an antibody against GFP. (F) WB analysis of D4a cells with BRG1 depleted using two shRNAs against BRG1 integrated into the genome. ( G ) Depleting BRG1 significantly impairs DNA DSB repair by HR, but not NHEJ. ( H ) The ratio of nucleosome density at 2.8 kb away from break sites at 8 h post I-SceI transfections vs before I-SceI transfections with or without BRG1 depletion. ( I ) Pretreatment with chloroquine rescues the decline of HR, but not NHEJ, in BRG1 depleted cells. ( J ) Epistasis analysis of PARP1 and BRG1 effect on nucleosome density. ( K ) Epistasis analysis of PARP1 and BRG1 effect on HR repair. All experiments were repeated at least three times. Error bars represent s.d. ** P

    Journal: Nucleic Acids Research

    Article Title: A PARP1–BRG1–SIRT1 axis promotes HR repair by reducing nucleosome density at DNA damage sites

    doi: 10.1093/nar/gkz592

    Figure Lengend Snippet: BRG1 recruited by PAR regulates chromatin relaxation to facilitate HR. ( A ) Co-IP and WB analysis of the interaction between BRG1 and PAR in response to IR. ( B ) ChIP analysis of BRG1 enrichment at DNA damage sites induced by I-SceI digestion in the presence or absence of the PARP1 inhibitor, olaparib. ( C ) Schematic representation of the BRG1 domain structure, and the truncated constructs used in this study. ( D ) Co-IP and WB analysis of the interaction between PAR and the three domains of BRG1 in the presence or absence of DNA DSBs induced by X-Ray. ( E ) Analysis of recruitment of three BRG1 domains to DSBs using ChIP assay. Different amounts of GFP tagged full length BRG1 and three BRG1 domains were transfected into NHEJ-I9A cells to ensure equal expression of them. Then ChIP was carried out by using an antibody against GFP. (F) WB analysis of D4a cells with BRG1 depleted using two shRNAs against BRG1 integrated into the genome. ( G ) Depleting BRG1 significantly impairs DNA DSB repair by HR, but not NHEJ. ( H ) The ratio of nucleosome density at 2.8 kb away from break sites at 8 h post I-SceI transfections vs before I-SceI transfections with or without BRG1 depletion. ( I ) Pretreatment with chloroquine rescues the decline of HR, but not NHEJ, in BRG1 depleted cells. ( J ) Epistasis analysis of PARP1 and BRG1 effect on nucleosome density. ( K ) Epistasis analysis of PARP1 and BRG1 effect on HR repair. All experiments were repeated at least three times. Error bars represent s.d. ** P

    Article Snippet: The antibodies used in the study are as follows: HA (Cell signaling, Cat. #2367), PARP1 (Cell signaling, Cat. #46D11), Actin (Santa cruz, Cat. #SC-47778), γH2Ax (Cell signaling, Cat. #9718S), CtIP (Active motif, Cat. #61141), Rad51(Abcam, Cat. #ab179897), His (Abways, Cat. #ab0002), BRG1 (Abcam, Cat. #ab70558), SIRT1 (Millipore, Cat. #07-131), PAR (Trevigen, Cat. #4335-MC-100), Flag (Abclonal, Cat. #AE005), AcK (Abcam, Cat. #ab21623).

    Techniques: Co-Immunoprecipitation Assay, Western Blot, Chromatin Immunoprecipitation, Construct, Transfection, Non-Homologous End Joining, Expressing

    In response to IR, SIRT1 deacetylates BRG1 to promote its ATPase activity, thereby stimulating nucleosome sliding to facilitate HR. ( A ) Analysis of the interaction between SIRT1 and BRG1 in response to IR. ( B ) Analysis of BRG1 acetylation levels in response to IR. ( C ) Co-IP analysis of BRG1 acetylation in 293T cells overexpressing SIRT1. ( D ) BRG1 is deacetylated by SIRT1 in vitro . The recombinant BRG1 was purified from 293F cells and incubated with recombinant SIRT1 for the deacetylation reaction. ( E ) BRG1 deacetylated by SIRT1 has higher ATPase activity than BRG1 with no SIRT1 treatment. The recombinant BRG1 was purified from 293F cells and incubated with recombinant SIRT1 before being subjected to the analysis of ATPase activity using the malachite green ATPase assay. ( F ) Co-IP analysis of acetylation levels of the three domains of BRG1. His-tagged three domains of BRG1 were transfected to 293F cells before co-IP and western blot analysis was performed. ( G ) Acetylation level of BRG1 ATPase domain WT and two mutants, K1029R and K1033R. His-tagged WT or mutated ATPase domain of BRG1 was transfected to 293T cells before co-IP and western blot analysis was performed. ( H ) Co-IP analysis of acetylation level of WT and the 2KR mutant containing both K1029R and K1033R mutations of BRG1-ATPase domain in 293T cells overexpressing SIRT1. ( I ) Acetylation level of purified recombinant BRG1 WT and the 2KR mutant. ( J ) Analysis of ATPase activity of BRG1 WT and 2KR mutant using the malachite green ATPase assay. ( K ) Analysis of nucleosome sliding activity of BRG1 WT and 2KR mutant in the presence of PARP1. ( L ) In both SIRT1 and BRG1 depleted cells only overexpressing BRG1 2KR mutant but not the WT or the ATPase dead mutant K785R rescues the impaired nucleosome clearance at DNA damage sites. ( M, N ) Quantification of RPA2 and RAD51 foci positive cells upon induction of DNA DSBs in both SIRT1 and BRG1 depleted cells. Cells transfected with different siRNA and expression vectors were irradiated on an X-Ray machine. At 4 h post IR, cells were fixed for immunofluorescence experiments. At least 50 Geminin positive cells were counted and only cells with over 10 RPA2 or RAD51 foci were counted as foci positive. In both SIRT1 and BRG1 depleting cells only overexpressing BRG1 2KR mutant but not the WT or the ATPase dead mutant K785R can rescue the impaired recruitment of RPA2 and RAD51 to DNA damage sites. ( O ) In SIRT1+ BRG1 depleted cells only overexpressing BRG1 2KR mutant, but not the WT BRG1 or the ATPase dead mutant K785R, partially rescues the reduced HR efficiency. All experiments were repeated at least three times. Error bars represent s.d. * P

    Journal: Nucleic Acids Research

    Article Title: A PARP1–BRG1–SIRT1 axis promotes HR repair by reducing nucleosome density at DNA damage sites

    doi: 10.1093/nar/gkz592

    Figure Lengend Snippet: In response to IR, SIRT1 deacetylates BRG1 to promote its ATPase activity, thereby stimulating nucleosome sliding to facilitate HR. ( A ) Analysis of the interaction between SIRT1 and BRG1 in response to IR. ( B ) Analysis of BRG1 acetylation levels in response to IR. ( C ) Co-IP analysis of BRG1 acetylation in 293T cells overexpressing SIRT1. ( D ) BRG1 is deacetylated by SIRT1 in vitro . The recombinant BRG1 was purified from 293F cells and incubated with recombinant SIRT1 for the deacetylation reaction. ( E ) BRG1 deacetylated by SIRT1 has higher ATPase activity than BRG1 with no SIRT1 treatment. The recombinant BRG1 was purified from 293F cells and incubated with recombinant SIRT1 before being subjected to the analysis of ATPase activity using the malachite green ATPase assay. ( F ) Co-IP analysis of acetylation levels of the three domains of BRG1. His-tagged three domains of BRG1 were transfected to 293F cells before co-IP and western blot analysis was performed. ( G ) Acetylation level of BRG1 ATPase domain WT and two mutants, K1029R and K1033R. His-tagged WT or mutated ATPase domain of BRG1 was transfected to 293T cells before co-IP and western blot analysis was performed. ( H ) Co-IP analysis of acetylation level of WT and the 2KR mutant containing both K1029R and K1033R mutations of BRG1-ATPase domain in 293T cells overexpressing SIRT1. ( I ) Acetylation level of purified recombinant BRG1 WT and the 2KR mutant. ( J ) Analysis of ATPase activity of BRG1 WT and 2KR mutant using the malachite green ATPase assay. ( K ) Analysis of nucleosome sliding activity of BRG1 WT and 2KR mutant in the presence of PARP1. ( L ) In both SIRT1 and BRG1 depleted cells only overexpressing BRG1 2KR mutant but not the WT or the ATPase dead mutant K785R rescues the impaired nucleosome clearance at DNA damage sites. ( M, N ) Quantification of RPA2 and RAD51 foci positive cells upon induction of DNA DSBs in both SIRT1 and BRG1 depleted cells. Cells transfected with different siRNA and expression vectors were irradiated on an X-Ray machine. At 4 h post IR, cells were fixed for immunofluorescence experiments. At least 50 Geminin positive cells were counted and only cells with over 10 RPA2 or RAD51 foci were counted as foci positive. In both SIRT1 and BRG1 depleting cells only overexpressing BRG1 2KR mutant but not the WT or the ATPase dead mutant K785R can rescue the impaired recruitment of RPA2 and RAD51 to DNA damage sites. ( O ) In SIRT1+ BRG1 depleted cells only overexpressing BRG1 2KR mutant, but not the WT BRG1 or the ATPase dead mutant K785R, partially rescues the reduced HR efficiency. All experiments were repeated at least three times. Error bars represent s.d. * P

    Article Snippet: The antibodies used in the study are as follows: HA (Cell signaling, Cat. #2367), PARP1 (Cell signaling, Cat. #46D11), Actin (Santa cruz, Cat. #SC-47778), γH2Ax (Cell signaling, Cat. #9718S), CtIP (Active motif, Cat. #61141), Rad51(Abcam, Cat. #ab179897), His (Abways, Cat. #ab0002), BRG1 (Abcam, Cat. #ab70558), SIRT1 (Millipore, Cat. #07-131), PAR (Trevigen, Cat. #4335-MC-100), Flag (Abclonal, Cat. #AE005), AcK (Abcam, Cat. #ab21623).

    Techniques: Activity Assay, Co-Immunoprecipitation Assay, In Vitro, Recombinant, Purification, Incubation, ATPase Assay, Transfection, Western Blot, Mutagenesis, Expressing, Irradiation, Immunofluorescence

    Effects of Brahma-related gene 1 (BRG1) on viability, apoptosis, and inflammatory factor expression in rheumatoid fibroblast-like synoviocyte MH7A cells. MH7A cells were transfected with pcDNA3.1-BRG1 vector to overexpress BRG1 or short hairpin RNA (shRNA) vector of BRG1 (sh-BRG1) to knock down BRG1. Blank vectors pcDNA3.1 and sh-control were transfected in the corresponding control group. (A) BRG1 inhibits cell viability as indicated by optical density (OD) at 570 nm. MTT was performed to detect cell viability at 0, 1, 2, and 3 d post-transfection. (B) BRG1 increases percent of apoptotic cells as quantified by flow cytometry at 48 h post-transfection. (C) BRG1 suppresses the mRNA level of inflammatory factors matrix metallopeptidase 3 ( MMP3 ), TIMP metallopeptidase inhibitor 2 ( TIMP2 ), cyclooxygenase 2 ( COX2 ), and interleukin 6 ( IL6 ) as revealed by qPCR at 48 h post-transfection. P values are indicated. BRG1 = Brahma-related gene 1, COX2 = cyclooxygenase 2, IL6 = interleukin 6, MMP3 = matrix metallopeptidase 3, MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide , OD = optical density, qPCR = quantitative polymerase chain reaction, shRNA = short hairpin RNA, TIMP2 = TIMP metallopeptidase inhibitor 2.

    Journal: Medicine

    Article Title: Brahma-related gene 1 induces apoptosis in a p53-dependent manner in human rheumatoid fibroblast-like synoviocyte MH7A

    doi: 10.1097/MD.0000000000005241

    Figure Lengend Snippet: Effects of Brahma-related gene 1 (BRG1) on viability, apoptosis, and inflammatory factor expression in rheumatoid fibroblast-like synoviocyte MH7A cells. MH7A cells were transfected with pcDNA3.1-BRG1 vector to overexpress BRG1 or short hairpin RNA (shRNA) vector of BRG1 (sh-BRG1) to knock down BRG1. Blank vectors pcDNA3.1 and sh-control were transfected in the corresponding control group. (A) BRG1 inhibits cell viability as indicated by optical density (OD) at 570 nm. MTT was performed to detect cell viability at 0, 1, 2, and 3 d post-transfection. (B) BRG1 increases percent of apoptotic cells as quantified by flow cytometry at 48 h post-transfection. (C) BRG1 suppresses the mRNA level of inflammatory factors matrix metallopeptidase 3 ( MMP3 ), TIMP metallopeptidase inhibitor 2 ( TIMP2 ), cyclooxygenase 2 ( COX2 ), and interleukin 6 ( IL6 ) as revealed by qPCR at 48 h post-transfection. P values are indicated. BRG1 = Brahma-related gene 1, COX2 = cyclooxygenase 2, IL6 = interleukin 6, MMP3 = matrix metallopeptidase 3, MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide , OD = optical density, qPCR = quantitative polymerase chain reaction, shRNA = short hairpin RNA, TIMP2 = TIMP metallopeptidase inhibitor 2.

    Article Snippet: The blot was incubated in the specific primary antibodies anti-BRG1, p53, proto-oncogene MDM2 (ab178938), cytochrome c (CYCS, ab133504), B-cell chronic lymphocytic leukemia (CLL)/lymphoma 2 (BCL2, ab32124), pro-caspase 3 (pro-CASP3, ab32150), cleaved CASP3 (ab32042), pro-CASP9 (ab69514), and cleaved CASP9 (ab2324, 1:1000, Abcam) overnight at 4°C.

    Techniques: Expressing, Transfection, Plasmid Preparation, shRNA, MTT Assay, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction

    Brahma-related gene 1 (BRG1) regulates rheumatoid fibroblast-like synoviocyte MH7A cell apoptosis in a p53-dependent manner. BRG1 interacts with p53, which can bind to the promoter region of apoptotic genes and regulate gene transcription. Altered apoptotic gene expression contributes to the regulation of cell apoptosis. BRG1 = Brahma-related gene 1.

    Journal: Medicine

    Article Title: Brahma-related gene 1 induces apoptosis in a p53-dependent manner in human rheumatoid fibroblast-like synoviocyte MH7A

    doi: 10.1097/MD.0000000000005241

    Figure Lengend Snippet: Brahma-related gene 1 (BRG1) regulates rheumatoid fibroblast-like synoviocyte MH7A cell apoptosis in a p53-dependent manner. BRG1 interacts with p53, which can bind to the promoter region of apoptotic genes and regulate gene transcription. Altered apoptotic gene expression contributes to the regulation of cell apoptosis. BRG1 = Brahma-related gene 1.

    Article Snippet: The blot was incubated in the specific primary antibodies anti-BRG1, p53, proto-oncogene MDM2 (ab178938), cytochrome c (CYCS, ab133504), B-cell chronic lymphocytic leukemia (CLL)/lymphoma 2 (BCL2, ab32124), pro-caspase 3 (pro-CASP3, ab32150), cleaved CASP3 (ab32042), pro-CASP9 (ab69514), and cleaved CASP9 (ab2324, 1:1000, Abcam) overnight at 4°C.

    Techniques: Expressing

    Overexpression and knockdown of Brahma-related gene 1 (BRG1) in rheumatoid fibroblast-like synoviocyte MH7A cells. MH7A cells were transfected with pcDNA3.1-BRG1 vector to overexpress BRG1 or short hairpin RNA (shRNA) vector of BRG1 (sh-BRG1) to knock down BRG1. Blank vectors pcDNA3.1 and sh-control were transfected in the corresponding control group. qPCR and Western blot were performed at 48 h post-transfection. GAPDH was used as an internal control. (A) The quantification of BRG1 mRNA level by qPCR in transfected cells. P values are indicated. (B) BRG1 protein level detected by Western blot in transfected cell. BRG1 = Brahma-related gene 1, IP = immunoprecipitation, GAPDH = glyceraldehyde 3-phosphate dehydrogenase, qPCR = quantitative polymerase chain reaction, shRNA = short hairpin RNA.

    Journal: Medicine

    Article Title: Brahma-related gene 1 induces apoptosis in a p53-dependent manner in human rheumatoid fibroblast-like synoviocyte MH7A

    doi: 10.1097/MD.0000000000005241

    Figure Lengend Snippet: Overexpression and knockdown of Brahma-related gene 1 (BRG1) in rheumatoid fibroblast-like synoviocyte MH7A cells. MH7A cells were transfected with pcDNA3.1-BRG1 vector to overexpress BRG1 or short hairpin RNA (shRNA) vector of BRG1 (sh-BRG1) to knock down BRG1. Blank vectors pcDNA3.1 and sh-control were transfected in the corresponding control group. qPCR and Western blot were performed at 48 h post-transfection. GAPDH was used as an internal control. (A) The quantification of BRG1 mRNA level by qPCR in transfected cells. P values are indicated. (B) BRG1 protein level detected by Western blot in transfected cell. BRG1 = Brahma-related gene 1, IP = immunoprecipitation, GAPDH = glyceraldehyde 3-phosphate dehydrogenase, qPCR = quantitative polymerase chain reaction, shRNA = short hairpin RNA.

    Article Snippet: The blot was incubated in the specific primary antibodies anti-BRG1, p53, proto-oncogene MDM2 (ab178938), cytochrome c (CYCS, ab133504), B-cell chronic lymphocytic leukemia (CLL)/lymphoma 2 (BCL2, ab32124), pro-caspase 3 (pro-CASP3, ab32150), cleaved CASP3 (ab32042), pro-CASP9 (ab69514), and cleaved CASP9 (ab2324, 1:1000, Abcam) overnight at 4°C.

    Techniques: Over Expression, Transfection, Plasmid Preparation, shRNA, Real-time Polymerase Chain Reaction, Western Blot, Immunoprecipitation

    Brahma-related gene 1 (BRG1) interacts with p53 and promotes p53 expression in rheumatoid fibroblast-like synoviocyte MH7A cells. MH7A cells were transfected with pcDNA3.1-BRG1 vector to overexpress BRG1. Blank vector pcDNA3.1 was transfected as a control. qPCR, immunoprecipitation (IP) and Western blot were performed at 48 h post-transfection, with GAPDH as an internal control. (A) BRG1 overexpression promotes p53 mRNA and inhibits proto-oncogene MDM2 mRNA as shown by qPCR results. P values are indicated. (B) BRG1 overexpression promotes protein levels of p53 and MDM2 as shown by qPCR results. (C) BRG1 interacts with p53 protein as revealed by IP. Cell lysates were immunoprecipitated with anti-p53 antibody (IP-p53) or anti-BRG1 antibody (IP-BRG1) and then BRG1 or p53 was detected by Western blot. Input, cell lysates without IP process is set as a positive control. IgG, IP with anti-immunoglobulin G (IgG) is set as a negative control. BRG1 = Brahma-related gene 1, IP = immunoprecipitation, MDM2 = murine double minute 2.

    Journal: Medicine

    Article Title: Brahma-related gene 1 induces apoptosis in a p53-dependent manner in human rheumatoid fibroblast-like synoviocyte MH7A

    doi: 10.1097/MD.0000000000005241

    Figure Lengend Snippet: Brahma-related gene 1 (BRG1) interacts with p53 and promotes p53 expression in rheumatoid fibroblast-like synoviocyte MH7A cells. MH7A cells were transfected with pcDNA3.1-BRG1 vector to overexpress BRG1. Blank vector pcDNA3.1 was transfected as a control. qPCR, immunoprecipitation (IP) and Western blot were performed at 48 h post-transfection, with GAPDH as an internal control. (A) BRG1 overexpression promotes p53 mRNA and inhibits proto-oncogene MDM2 mRNA as shown by qPCR results. P values are indicated. (B) BRG1 overexpression promotes protein levels of p53 and MDM2 as shown by qPCR results. (C) BRG1 interacts with p53 protein as revealed by IP. Cell lysates were immunoprecipitated with anti-p53 antibody (IP-p53) or anti-BRG1 antibody (IP-BRG1) and then BRG1 or p53 was detected by Western blot. Input, cell lysates without IP process is set as a positive control. IgG, IP with anti-immunoglobulin G (IgG) is set as a negative control. BRG1 = Brahma-related gene 1, IP = immunoprecipitation, MDM2 = murine double minute 2.

    Article Snippet: The blot was incubated in the specific primary antibodies anti-BRG1, p53, proto-oncogene MDM2 (ab178938), cytochrome c (CYCS, ab133504), B-cell chronic lymphocytic leukemia (CLL)/lymphoma 2 (BCL2, ab32124), pro-caspase 3 (pro-CASP3, ab32150), cleaved CASP3 (ab32042), pro-CASP9 (ab69514), and cleaved CASP9 (ab2324, 1:1000, Abcam) overnight at 4°C.

    Techniques: Expressing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Western Blot, Over Expression, Positive Control, Negative Control

    Regulation of apoptotic factors by BRG1 depends on p53. Rheumatoid fibroblast-like synoviocyte MH7A cells were transfected with pcDNA3.1-BRG1 vector to overexpress BRG1, or the short hairpin RNA for p53 (sh-p53) to knockdown p53. Western blot was performed at 48 h post-transfection. (A) Western blot showing that apoptotic factors cytochrome c (CYCS), cleaved caspase 3 (cleaved-CASP3), cleaved-CASP9, and B-cell CLL/lymphoma 2 (BCL2) were regulated by BRG1, but the regulation was suppressed when p53 was knocked down. (B) Relative protein levels calculated based on the band density in Western blot results. P values are indicated. BCL2 = B-cell chronic lymphocytic leukemia/lymphoma 2, BRG1 = Brahma-related gene 1, cleaved-CASP3 = cleaved caspase 3, CYCS = cytochrome c.

    Journal: Medicine

    Article Title: Brahma-related gene 1 induces apoptosis in a p53-dependent manner in human rheumatoid fibroblast-like synoviocyte MH7A

    doi: 10.1097/MD.0000000000005241

    Figure Lengend Snippet: Regulation of apoptotic factors by BRG1 depends on p53. Rheumatoid fibroblast-like synoviocyte MH7A cells were transfected with pcDNA3.1-BRG1 vector to overexpress BRG1, or the short hairpin RNA for p53 (sh-p53) to knockdown p53. Western blot was performed at 48 h post-transfection. (A) Western blot showing that apoptotic factors cytochrome c (CYCS), cleaved caspase 3 (cleaved-CASP3), cleaved-CASP9, and B-cell CLL/lymphoma 2 (BCL2) were regulated by BRG1, but the regulation was suppressed when p53 was knocked down. (B) Relative protein levels calculated based on the band density in Western blot results. P values are indicated. BCL2 = B-cell chronic lymphocytic leukemia/lymphoma 2, BRG1 = Brahma-related gene 1, cleaved-CASP3 = cleaved caspase 3, CYCS = cytochrome c.

    Article Snippet: The blot was incubated in the specific primary antibodies anti-BRG1, p53, proto-oncogene MDM2 (ab178938), cytochrome c (CYCS, ab133504), B-cell chronic lymphocytic leukemia (CLL)/lymphoma 2 (BCL2, ab32124), pro-caspase 3 (pro-CASP3, ab32150), cleaved CASP3 (ab32042), pro-CASP9 (ab69514), and cleaved CASP9 (ab2324, 1:1000, Abcam) overnight at 4°C.

    Techniques: Transfection, Plasmid Preparation, shRNA, Western Blot

    Withdrawal from cocaine self-administration increases functional BRG1—SMAD3 interaction ( A ) Timeline of behavioral testing and tissue collection. ( B ) Mean number of saline and cocaine (1 mg/kg/inf) infusions per session during self-administration training ( n = 19–22/group). ( C ) Protein lysates from rat nucleus accumbens (NAc) and caudoputamen (CPU) collected following withdrawal from cocaine or saline self-administration were immunoprecipitated with anti-BRG1 antibody and probed for SMAD3 binding ( n = 5–8/group). ( D ) Occupancy of BRG1 on the promoter regions of β-catenin ( Ctnnb1 ), NMDA receptor 2A ( Grin2a ), myocyte enhancer factor 2d ( Mef2d ), adenylyl cyclase-associated protein 2 ( Cap2 ), drebrin ( Dbn1 ), and prodynorphin ( Pdyn ) in the NAc as measured by quantitative chromatin immunoprecipitation following 7-d withdrawal from saline or cocaine self-administration ( n = 5–7 samples/group (two animals count as one sample)). Data are expressed as mean ± SEM; * P

    Journal: Biological psychiatry

    Article Title: BRG1 in the nucleus accumbens regulates cocaine-seeking behavior

    doi: 10.1016/j.biopsych.2016.04.020

    Figure Lengend Snippet: Withdrawal from cocaine self-administration increases functional BRG1—SMAD3 interaction ( A ) Timeline of behavioral testing and tissue collection. ( B ) Mean number of saline and cocaine (1 mg/kg/inf) infusions per session during self-administration training ( n = 19–22/group). ( C ) Protein lysates from rat nucleus accumbens (NAc) and caudoputamen (CPU) collected following withdrawal from cocaine or saline self-administration were immunoprecipitated with anti-BRG1 antibody and probed for SMAD3 binding ( n = 5–8/group). ( D ) Occupancy of BRG1 on the promoter regions of β-catenin ( Ctnnb1 ), NMDA receptor 2A ( Grin2a ), myocyte enhancer factor 2d ( Mef2d ), adenylyl cyclase-associated protein 2 ( Cap2 ), drebrin ( Dbn1 ), and prodynorphin ( Pdyn ) in the NAc as measured by quantitative chromatin immunoprecipitation following 7-d withdrawal from saline or cocaine self-administration ( n = 5–7 samples/group (two animals count as one sample)). Data are expressed as mean ± SEM; * P

    Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies diluted in blocking buffer (Rockland Immunochemicals, Inc., Limerick, PA), including: rabbit anti-BRG1 (1:2,000; Abcam, Cambridge, MA), rabbit anti-phospho(p)-SMAD3 (1:500; Calbiochem of Millipore Corp., Billerica, MA), and mouse anti-β-actin (1:10,000; Cell Signaling Technologies, Inc., Danvers, MA).

    Techniques: Functional Assay, Immunoprecipitation, Binding Assay, Chromatin Immunoprecipitation

    Withdrawal from cocaine self-administration increases p-SMAD3 and BRG1 protein levels and interaction ( A ) Timeline of behavioral testing and tissue collection. ( B ) Mean number of saline and cocaine (1 mg/kg/inf) infusions per session during self-administration training ( n = 6/group). ( C ) Representative anatomical location of tissue punches taken from rat nucleus accumbens (NAc) and caudoputamen (CPU). ( D – G ) Relative p-SMAD3 and BRG1 expression in the NAc ( D, E ; n = 6/group) and CPU ( F, G; n = 5–6/group). Data are expressed as mean ± SEM; * P

    Journal: Biological psychiatry

    Article Title: BRG1 in the nucleus accumbens regulates cocaine-seeking behavior

    doi: 10.1016/j.biopsych.2016.04.020

    Figure Lengend Snippet: Withdrawal from cocaine self-administration increases p-SMAD3 and BRG1 protein levels and interaction ( A ) Timeline of behavioral testing and tissue collection. ( B ) Mean number of saline and cocaine (1 mg/kg/inf) infusions per session during self-administration training ( n = 6/group). ( C ) Representative anatomical location of tissue punches taken from rat nucleus accumbens (NAc) and caudoputamen (CPU). ( D – G ) Relative p-SMAD3 and BRG1 expression in the NAc ( D, E ; n = 6/group) and CPU ( F, G; n = 5–6/group). Data are expressed as mean ± SEM; * P

    Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies diluted in blocking buffer (Rockland Immunochemicals, Inc., Limerick, PA), including: rabbit anti-BRG1 (1:2,000; Abcam, Cambridge, MA), rabbit anti-phospho(p)-SMAD3 (1:500; Calbiochem of Millipore Corp., Billerica, MA), and mouse anti-β-actin (1:10,000; Cell Signaling Technologies, Inc., Danvers, MA).

    Techniques: Expressing

    Pharmacological inhibition of BRG1 in the nucleus accumbens decreases cue-induced reinstatement of cocaine seeking ( A ) Timeline of behavioral testing and intra-accumbal microinjections. ( B ) Mean number of infusions per session during cocaine self-administration (1 mg/kg/inf) and ( C ) total responses during extinction did not differ between groups assigned to receive BRG1 inhibitor PFI3 or vehicle. ( D ) Number of active responses during cue-induced reinstatement following microinjection (1 μL/hemisphere) of vehicle or PFI3 (30 mM) ( n = 7–8/group). ( E ) SMAD3 and BRG1 co-immunoprecipitation following microinjection of vehicle or PFI3 ( n = 6/group). ( F ) Total distance traveled in a locomotor test and ( G ) responding for a food reward did not differ between groups ( n = 7–8/group). Data are expressed as mean ± SEM; * P

    Journal: Biological psychiatry

    Article Title: BRG1 in the nucleus accumbens regulates cocaine-seeking behavior

    doi: 10.1016/j.biopsych.2016.04.020

    Figure Lengend Snippet: Pharmacological inhibition of BRG1 in the nucleus accumbens decreases cue-induced reinstatement of cocaine seeking ( A ) Timeline of behavioral testing and intra-accumbal microinjections. ( B ) Mean number of infusions per session during cocaine self-administration (1 mg/kg/inf) and ( C ) total responses during extinction did not differ between groups assigned to receive BRG1 inhibitor PFI3 or vehicle. ( D ) Number of active responses during cue-induced reinstatement following microinjection (1 μL/hemisphere) of vehicle or PFI3 (30 mM) ( n = 7–8/group). ( E ) SMAD3 and BRG1 co-immunoprecipitation following microinjection of vehicle or PFI3 ( n = 6/group). ( F ) Total distance traveled in a locomotor test and ( G ) responding for a food reward did not differ between groups ( n = 7–8/group). Data are expressed as mean ± SEM; * P

    Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies diluted in blocking buffer (Rockland Immunochemicals, Inc., Limerick, PA), including: rabbit anti-BRG1 (1:2,000; Abcam, Cambridge, MA), rabbit anti-phospho(p)-SMAD3 (1:500; Calbiochem of Millipore Corp., Billerica, MA), and mouse anti-β-actin (1:10,000; Cell Signaling Technologies, Inc., Danvers, MA).

    Techniques: Inhibition, Immunoprecipitation

    Overexpression of Brg1 in the nucleus accumbens enhances cue-induced reinstatement of cocaine seeking ( A ) Timeline of behavioral testing and intra-accumbal virus injection. ( B ) Representative picture of a coronal section of the rat brain (1.7 mm from bregma), depicting GFP-infected cells in the nucleus accumbens (AC: anterior commissure). Representative Western blots showing increased BRG1 protein levels in the nucleus accumbens following HSV -Brg1 overexpression. ( C ) Mean number of infusions per session during cocaine self-administration (1 mg/kg/inf) and ( D ) total responses during extinction procedure did not differ between groups assigned to receive HSV- Brg1 or HSV-GFP. ( E ) Number of active responses during cue-induced reinstatement following overexpression of HSV-GFP or HSV- Brg1 ( n = 8–10/group). ( F ) Total distance traveled in a locomotor test ( n = 8/group). Data are expressed as mean ± SEM, * P

    Journal: Biological psychiatry

    Article Title: BRG1 in the nucleus accumbens regulates cocaine-seeking behavior

    doi: 10.1016/j.biopsych.2016.04.020

    Figure Lengend Snippet: Overexpression of Brg1 in the nucleus accumbens enhances cue-induced reinstatement of cocaine seeking ( A ) Timeline of behavioral testing and intra-accumbal virus injection. ( B ) Representative picture of a coronal section of the rat brain (1.7 mm from bregma), depicting GFP-infected cells in the nucleus accumbens (AC: anterior commissure). Representative Western blots showing increased BRG1 protein levels in the nucleus accumbens following HSV -Brg1 overexpression. ( C ) Mean number of infusions per session during cocaine self-administration (1 mg/kg/inf) and ( D ) total responses during extinction procedure did not differ between groups assigned to receive HSV- Brg1 or HSV-GFP. ( E ) Number of active responses during cue-induced reinstatement following overexpression of HSV-GFP or HSV- Brg1 ( n = 8–10/group). ( F ) Total distance traveled in a locomotor test ( n = 8/group). Data are expressed as mean ± SEM, * P

    Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies diluted in blocking buffer (Rockland Immunochemicals, Inc., Limerick, PA), including: rabbit anti-BRG1 (1:2,000; Abcam, Cambridge, MA), rabbit anti-phospho(p)-SMAD3 (1:500; Calbiochem of Millipore Corp., Billerica, MA), and mouse anti-β-actin (1:10,000; Cell Signaling Technologies, Inc., Danvers, MA).

    Techniques: Over Expression, Injection, Infection, Western Blot

    The role of Brg1 in AML12 cells subjected to H/R injury. ( a ) Western blot analysis showed the cellular Brg1 and HO-1 protein expression in AML12 hepatocytes subjected to hypoxia for 12 h and reoxygenation for 0 (H12R0), 2 (H12R2), 4 (H12R4), 6 (H12R6), 8 (H12R8), 12 (H12R12), 16 (H12R16) and 24 h (H12R24) before sample collection in comparison with the cells cultured for the same time as control (namely, C12, C14, C16, C20, C24, C28 and C36, representing 12–36 h of culture). The proteins from the H/R groups and from the control groups were loaded in the same gel when performing western blotting assay and displayed in parallel to facilitate comparison. Representative images from one of three independent experiments were shown. ( b and c ) Quantitative measurement of band intensity in a by densitometry analysis. ( d ) Fluorescence immunostaining of DCF in cells with Brg1 overexpression using Brg1-Adv transfection, and elevated in cells with Brg1 knockdown using Brg1-siRNA transfection during H/R (H12R4) injury. Representative images from one of three independent experiments were shown. ( e ) Bar graph showing the change in DCF fluorescent intensity. ( f ) ELISA assay showed that 8-isoprostane level was decreased after Brg1-Adv treatment and increased by Brg1-siRNA transfection during H/R (H12R4) injury. ( g and h ) Western blot analysis showed the change of Brg1 and Nrf2 protein expression in AML12 cells, respectively, under condition of Brg1 overexpression or knockdown. Representative images were shown and quantitative measurements were performed. ( i and j ) Western blot and RT-PCR analysis showed the protein and mRNA level of HO-1 and NQO1 in response to Brg1 overexpression or knockdown during cell H/R (H12R4) injury. ( k ) HO-1 promoter-driven luciferase activity assay was performed and tBHQ (20 μ M) was used as Nrf2 nuclear translocation positive control. Data are mean±S.E.M. of three independent experiments each performed in triplicate. * P

    Journal: Cell Death & Disease

    Article Title: Brg1-mediated Nrf2/HO-1 pathway activation alleviates hepatic ischemia–reperfusion injury

    doi: 10.1038/cddis.2017.236

    Figure Lengend Snippet: The role of Brg1 in AML12 cells subjected to H/R injury. ( a ) Western blot analysis showed the cellular Brg1 and HO-1 protein expression in AML12 hepatocytes subjected to hypoxia for 12 h and reoxygenation for 0 (H12R0), 2 (H12R2), 4 (H12R4), 6 (H12R6), 8 (H12R8), 12 (H12R12), 16 (H12R16) and 24 h (H12R24) before sample collection in comparison with the cells cultured for the same time as control (namely, C12, C14, C16, C20, C24, C28 and C36, representing 12–36 h of culture). The proteins from the H/R groups and from the control groups were loaded in the same gel when performing western blotting assay and displayed in parallel to facilitate comparison. Representative images from one of three independent experiments were shown. ( b and c ) Quantitative measurement of band intensity in a by densitometry analysis. ( d ) Fluorescence immunostaining of DCF in cells with Brg1 overexpression using Brg1-Adv transfection, and elevated in cells with Brg1 knockdown using Brg1-siRNA transfection during H/R (H12R4) injury. Representative images from one of three independent experiments were shown. ( e ) Bar graph showing the change in DCF fluorescent intensity. ( f ) ELISA assay showed that 8-isoprostane level was decreased after Brg1-Adv treatment and increased by Brg1-siRNA transfection during H/R (H12R4) injury. ( g and h ) Western blot analysis showed the change of Brg1 and Nrf2 protein expression in AML12 cells, respectively, under condition of Brg1 overexpression or knockdown. Representative images were shown and quantitative measurements were performed. ( i and j ) Western blot and RT-PCR analysis showed the protein and mRNA level of HO-1 and NQO1 in response to Brg1 overexpression or knockdown during cell H/R (H12R4) injury. ( k ) HO-1 promoter-driven luciferase activity assay was performed and tBHQ (20 μ M) was used as Nrf2 nuclear translocation positive control. Data are mean±S.E.M. of three independent experiments each performed in triplicate. * P

    Article Snippet: Antibodies and reagents Antibodies recognizing Brg1 and Nrf2 were purchased from Abcam Company (Cambridge, MA, USA).

    Techniques: Western Blot, Expressing, Cell Culture, Fluorescence, Immunostaining, Over Expression, Transfection, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Luciferase, Activity Assay, Translocation Assay, Positive Control

    Inhibition of HO-1 reversed the protective effects of Brg1 overexpression. CMV-Brg1 transgenic mice subjected to HIR with or without HO-1 inhibitor ZnPP. Animals were killed at 6 h after reperfusion onset. ( a ) Liver pathology was detected by H E staining and HO-1 expression was measured by immunohistochemical staining. Representative images from one of three independent experiments were shown. ( b ) The effects of HO-1 inhibition on Brg1-CMV mice post-HIR liver function were examined by AST and ALT. ( c ) Quantitative measurement of HO-1 immunohistochemical staining density in a by densitometry analysis. ( d and e ) Furthermore, AML12 cells injury was attenuated by overexpression of HO-1 in hepatocytes subjected to H/R (H12R4). Cell DCF fluorescence was detected and relative DCF fluorescence intensity was assayed. Representative images from one of three independent experiments were shown. ( f ) Cell 8-isoprostane was detected by ELISA assay to show the cellular oxidative stress level. Data are mean±S.E.M. of three independent experiments each performed in triplicate. * P

    Journal: Cell Death & Disease

    Article Title: Brg1-mediated Nrf2/HO-1 pathway activation alleviates hepatic ischemia–reperfusion injury

    doi: 10.1038/cddis.2017.236

    Figure Lengend Snippet: Inhibition of HO-1 reversed the protective effects of Brg1 overexpression. CMV-Brg1 transgenic mice subjected to HIR with or without HO-1 inhibitor ZnPP. Animals were killed at 6 h after reperfusion onset. ( a ) Liver pathology was detected by H E staining and HO-1 expression was measured by immunohistochemical staining. Representative images from one of three independent experiments were shown. ( b ) The effects of HO-1 inhibition on Brg1-CMV mice post-HIR liver function were examined by AST and ALT. ( c ) Quantitative measurement of HO-1 immunohistochemical staining density in a by densitometry analysis. ( d and e ) Furthermore, AML12 cells injury was attenuated by overexpression of HO-1 in hepatocytes subjected to H/R (H12R4). Cell DCF fluorescence was detected and relative DCF fluorescence intensity was assayed. Representative images from one of three independent experiments were shown. ( f ) Cell 8-isoprostane was detected by ELISA assay to show the cellular oxidative stress level. Data are mean±S.E.M. of three independent experiments each performed in triplicate. * P

    Article Snippet: Antibodies and reagents Antibodies recognizing Brg1 and Nrf2 were purchased from Abcam Company (Cambridge, MA, USA).

    Techniques: Inhibition, Over Expression, Transgenic Assay, Mouse Assay, Staining, Expressing, Immunohistochemistry, AST Assay, Fluorescence, Enzyme-linked Immunosorbent Assay

    Expressions of Brg1, Nrf2 and Nrf2 downstream genes in the liver after hepatic I/R. ( a , b , c and d ) Western blot analysis showed that Brg1, nuclear Nrf2, HO-1 and NQO1 protein expressions were elevated in response to HIR in the liver at indicated time points. Representative images from one of three independent experiments were shown. Quantitative analyses of the results were also performed. ( e , f , g and h ) Transcript levels of Brg1, Nrf2, HO-1 and NQO1 in the liver in sham and HIR group were measured by RT-PCR. Each bar represents the mean±S.E.M. ( n=6 per group). * P

    Journal: Cell Death & Disease

    Article Title: Brg1-mediated Nrf2/HO-1 pathway activation alleviates hepatic ischemia–reperfusion injury

    doi: 10.1038/cddis.2017.236

    Figure Lengend Snippet: Expressions of Brg1, Nrf2 and Nrf2 downstream genes in the liver after hepatic I/R. ( a , b , c and d ) Western blot analysis showed that Brg1, nuclear Nrf2, HO-1 and NQO1 protein expressions were elevated in response to HIR in the liver at indicated time points. Representative images from one of three independent experiments were shown. Quantitative analyses of the results were also performed. ( e , f , g and h ) Transcript levels of Brg1, Nrf2, HO-1 and NQO1 in the liver in sham and HIR group were measured by RT-PCR. Each bar represents the mean±S.E.M. ( n=6 per group). * P

    Article Snippet: Antibodies and reagents Antibodies recognizing Brg1 and Nrf2 were purchased from Abcam Company (Cambridge, MA, USA).

    Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction

    Proposed signaling of Brg1-mediated Nrf2/HO-1 pathway activation in HIR injury. Keap1 dissociation from Nrf2 leads to Nrf2 translocation from cytoplasm to nucleus during HIR injury. However, with poor efficiency, hepatocyte could not produce enough antioxidant HO-1, which is the downstream target gene of keap1/Nrf2 pathway. Upregulation of the chromatin remodeling factor Brg1 could enhance transcription factor Nrf2 binding to HO-1 DNA sequence and promote HO-1 generation in a high efficiency way. Antioxidant enzyme HO-1 then suppresses the free radical generated from oxidative burst during hepatocyte H/R injury

    Journal: Cell Death & Disease

    Article Title: Brg1-mediated Nrf2/HO-1 pathway activation alleviates hepatic ischemia–reperfusion injury

    doi: 10.1038/cddis.2017.236

    Figure Lengend Snippet: Proposed signaling of Brg1-mediated Nrf2/HO-1 pathway activation in HIR injury. Keap1 dissociation from Nrf2 leads to Nrf2 translocation from cytoplasm to nucleus during HIR injury. However, with poor efficiency, hepatocyte could not produce enough antioxidant HO-1, which is the downstream target gene of keap1/Nrf2 pathway. Upregulation of the chromatin remodeling factor Brg1 could enhance transcription factor Nrf2 binding to HO-1 DNA sequence and promote HO-1 generation in a high efficiency way. Antioxidant enzyme HO-1 then suppresses the free radical generated from oxidative burst during hepatocyte H/R injury

    Article Snippet: Antibodies and reagents Antibodies recognizing Brg1 and Nrf2 were purchased from Abcam Company (Cambridge, MA, USA).

    Techniques: Activation Assay, Translocation Assay, Binding Assay, Sequencing, Generated

    Overexpression of Brg1 attenuated HIR injury via enhancing antioxidant enzyme. Animals were subjected to 70% liver warm ischemia for 60 min and live tissues were collected at 6 h after reperfusion. ( a ) Western blot analysis showed that Brg1 expression was increased in Brg1 overexpression (CMV-Brg1) mice compared to WT mice both in the sham and HIR groups. ( b ) Suzike’s injury score showed lower scores in CMV-Brg1 mice than in WT mice after HIR injury. ( c ) Representative H E staining images of liver collected from WT and CMV-Brg1 mice in the sham and HIR groups are shown. ( d and e ) Serum AST and ALT concentration showed an improved liver function in CMV-Brg1 mice after HIR injury. ( f ) ELISA analysis showed elevation of 8-isoprostane level was attenuated in CMV-Brg1 mice in response to HIR injury relative to that in the control. ( g ) ROS production measured by fluorescence intensity of DCF was reduced in CMV-Brg1 mice after HIR injury. ( h ) Immumohistochemical staining showed that liver HO-1 protein expression was elevated in response to HIR in CMV-Brg1 mice. ( i ) Quantitative analyses of the results from h were also performed. ( j ) Western blot analysis showed that liver NQO1 protein expression did not significantly change in CMV-Brg1 mice compared to WT mice. Each bar represents the mean±S.E.M. ( n=6 per group). * P

    Journal: Cell Death & Disease

    Article Title: Brg1-mediated Nrf2/HO-1 pathway activation alleviates hepatic ischemia–reperfusion injury

    doi: 10.1038/cddis.2017.236

    Figure Lengend Snippet: Overexpression of Brg1 attenuated HIR injury via enhancing antioxidant enzyme. Animals were subjected to 70% liver warm ischemia for 60 min and live tissues were collected at 6 h after reperfusion. ( a ) Western blot analysis showed that Brg1 expression was increased in Brg1 overexpression (CMV-Brg1) mice compared to WT mice both in the sham and HIR groups. ( b ) Suzike’s injury score showed lower scores in CMV-Brg1 mice than in WT mice after HIR injury. ( c ) Representative H E staining images of liver collected from WT and CMV-Brg1 mice in the sham and HIR groups are shown. ( d and e ) Serum AST and ALT concentration showed an improved liver function in CMV-Brg1 mice after HIR injury. ( f ) ELISA analysis showed elevation of 8-isoprostane level was attenuated in CMV-Brg1 mice in response to HIR injury relative to that in the control. ( g ) ROS production measured by fluorescence intensity of DCF was reduced in CMV-Brg1 mice after HIR injury. ( h ) Immumohistochemical staining showed that liver HO-1 protein expression was elevated in response to HIR in CMV-Brg1 mice. ( i ) Quantitative analyses of the results from h were also performed. ( j ) Western blot analysis showed that liver NQO1 protein expression did not significantly change in CMV-Brg1 mice compared to WT mice. Each bar represents the mean±S.E.M. ( n=6 per group). * P

    Article Snippet: Antibodies and reagents Antibodies recognizing Brg1 and Nrf2 were purchased from Abcam Company (Cambridge, MA, USA).

    Techniques: Over Expression, Western Blot, Expressing, Mouse Assay, Staining, AST Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Fluorescence

    HO-1 promoter was regulated by Brg1/Nrf2 upon hepatocytes H/R. ( a ) AML12 cells were transfected with PGL3-HO-1-Luc, Brg1-Adv expression plasmids, Neh4 and/or Neh5 Nrf2 deletion mutants (△Neh4/△Neh5) without or with hypoxia for 12 h and reoxygenation for 4 h. Transfections and HO-1 promoter-driven luciferase assays were performed and tBHQ (20 μ M) was used as Nrf2 nuclear translocation positive control. ( b ) AML12 hepatocytes were then pretreated without or with Brg1-siRNA, or Brg1-Adv and then subjected to hypoxia for 12 h and reoxygenation for 4 h before sample collection. ChIP analyses were performed with antibodies against Brg1 and primers for the HO-1 promoter regions. ( c and d ) Furthermore, hepatocytes were pretreated without or with Nrf2 siRNA and Brg-Adv, then subjected to hypoxia for 12 h and reoxygenation for 4 h, Co-IP analysis were also performed with antibody against Nrf2. IgG was used as a negative control. Quantitative measurement of Brg1 band intensity was performed by densitometry analysis. ( e ) Diagram of HO-1 promoter activated by Brg1/Nrf2 upon H/R. Both human and mouse HO-1 genes have two important distal enhancer regions, E1 and E2, located about 4 and 10 kbp upstream of the transcription start site. The dominant element in the E1 and E2 regions is the ARE, which mediates transcriptional activation in response to almost all HO-1 inducers tested. ARE represent binding sites of several transcription factors such as Nrf2. Under HIR condition, nuclear Brg1 interacts with Nrf2 via transactivation domain, Nrf2 ECH homology (Neh)4 and Neh5, which promotes Nrf2 binding to the ARE within the gene promoter of HO-1. Data are mean±S.E.M. of three independent experiments each performed in triplicate. * P

    Journal: Cell Death & Disease

    Article Title: Brg1-mediated Nrf2/HO-1 pathway activation alleviates hepatic ischemia–reperfusion injury

    doi: 10.1038/cddis.2017.236

    Figure Lengend Snippet: HO-1 promoter was regulated by Brg1/Nrf2 upon hepatocytes H/R. ( a ) AML12 cells were transfected with PGL3-HO-1-Luc, Brg1-Adv expression plasmids, Neh4 and/or Neh5 Nrf2 deletion mutants (△Neh4/△Neh5) without or with hypoxia for 12 h and reoxygenation for 4 h. Transfections and HO-1 promoter-driven luciferase assays were performed and tBHQ (20 μ M) was used as Nrf2 nuclear translocation positive control. ( b ) AML12 hepatocytes were then pretreated without or with Brg1-siRNA, or Brg1-Adv and then subjected to hypoxia for 12 h and reoxygenation for 4 h before sample collection. ChIP analyses were performed with antibodies against Brg1 and primers for the HO-1 promoter regions. ( c and d ) Furthermore, hepatocytes were pretreated without or with Nrf2 siRNA and Brg-Adv, then subjected to hypoxia for 12 h and reoxygenation for 4 h, Co-IP analysis were also performed with antibody against Nrf2. IgG was used as a negative control. Quantitative measurement of Brg1 band intensity was performed by densitometry analysis. ( e ) Diagram of HO-1 promoter activated by Brg1/Nrf2 upon H/R. Both human and mouse HO-1 genes have two important distal enhancer regions, E1 and E2, located about 4 and 10 kbp upstream of the transcription start site. The dominant element in the E1 and E2 regions is the ARE, which mediates transcriptional activation in response to almost all HO-1 inducers tested. ARE represent binding sites of several transcription factors such as Nrf2. Under HIR condition, nuclear Brg1 interacts with Nrf2 via transactivation domain, Nrf2 ECH homology (Neh)4 and Neh5, which promotes Nrf2 binding to the ARE within the gene promoter of HO-1. Data are mean±S.E.M. of three independent experiments each performed in triplicate. * P

    Article Snippet: Antibodies and reagents Antibodies recognizing Brg1 and Nrf2 were purchased from Abcam Company (Cambridge, MA, USA).

    Techniques: Transfection, Expressing, Luciferase, Translocation Assay, Positive Control, Chromatin Immunoprecipitation, Co-Immunoprecipitation Assay, Negative Control, Activation Assay, Binding Assay

    ARID1A loss impairs enhancer-mediated gene regulation in the colonic epithelium a) Observed vs. expected TF motif instances at TSS-distal H3K27ac regions (enhancers) with reduced H3K27ac signal based on enrichment regions with stable H3K27ac signal. Motifs highly similar to AP1 and CTCF motifs are highlighted; b) Correlation between H3K27ac signal change ( Arid1a −/− / WT) and WT ChIP-Seq signal levels of different factors profiled in this work and by the ENCODE Project. c) ChIP-Seq profiles for SMARCA4, SMARCC1, JUND, FOSL1, and CTCF in WT HCT116 cells centered around TSS-distal H3K27ac regions (enhancers) that remain stable, show lost/weakened H3K27ac, or show gained/strengthened H3K27ac in Arid1a −/− cells relative to WT; d) H3K27ac levels at TSS-proximal (promoter) and TSS-distal (enhancer) enrichment regions for colon epithelium from wildtype (WT) and Villin-Cre ER-T2 Arid1a fl/fl ( Arid1a −/− ) mice (Note: numbers in the three corners denote numbers of activated ( > 2×), inactivated (

    Journal: Nature genetics

    Article Title: ARID1A loss impairs enhancer-mediated gene regulation and drives colon cancer in mice

    doi: 10.1038/ng.3744

    Figure Lengend Snippet: ARID1A loss impairs enhancer-mediated gene regulation in the colonic epithelium a) Observed vs. expected TF motif instances at TSS-distal H3K27ac regions (enhancers) with reduced H3K27ac signal based on enrichment regions with stable H3K27ac signal. Motifs highly similar to AP1 and CTCF motifs are highlighted; b) Correlation between H3K27ac signal change ( Arid1a −/− / WT) and WT ChIP-Seq signal levels of different factors profiled in this work and by the ENCODE Project. c) ChIP-Seq profiles for SMARCA4, SMARCC1, JUND, FOSL1, and CTCF in WT HCT116 cells centered around TSS-distal H3K27ac regions (enhancers) that remain stable, show lost/weakened H3K27ac, or show gained/strengthened H3K27ac in Arid1a −/− cells relative to WT; d) H3K27ac levels at TSS-proximal (promoter) and TSS-distal (enhancer) enrichment regions for colon epithelium from wildtype (WT) and Villin-Cre ER-T2 Arid1a fl/fl ( Arid1a −/− ) mice (Note: numbers in the three corners denote numbers of activated ( > 2×), inactivated (

    Article Snippet: The following antibodies were used for immunoprecipitation of 30ug solubilized chromatin: SMARCC1/BAF155 (Santa Cruz sc9746; ChIP-Seq and ChIP-qPCR), SMARCA4/BRG1 (Abcam ab110641; ChIP-Seq and ChIP-qPCR), H3K27ac (Abcam ab4729; ChIP-Seq and ChIP-qPCR); H3K4me (Abcam ab8895; ChIP-Seq), H3K4me3 (Abcam ab8580; ChIP-Seq); ARID1A (Santa Cruz 32761 and Millipore PSG3; ChIP-qPCR) and ARID1B (Abcam ab57461 and Santa Cruz 32762; ChIP-qPCR).

    Techniques: Chromatin Immunoprecipitation, Mouse Assay

    ARID1A loss causes defects in SWI/SNF targeting to chromatin a) Live cell morphology of HCT116 ARID1A WT, ARID1A +/− , and Arid1a −/− cells in culture; b) Proliferation measured by MTT assay, error bars show standard deviation of 3 measurements (technical replicates); c) Invasion measured by Matrigel-chamber based assay, error bars show standard deviation of 4 measurements (technical replicates); d) Protein levels of ARID1A, E-Cadherin, and β-actin; e) Protein levels of Vimentin and β-actin (Note: NIH 3T3 fibroblast cells included for positive control); f) Fold change (log 2 ) in SMARCA4 and SMARCC1 ChIP-Seq signals at SWI/SNF binding sites in Arid1a −/− cells relative to WT; g) Immunoprecipitation of SWI/SNF complexes using antibodies targeting ARID1A and ARID1B; h) Protein levels of ARID1B and β-actin following ARID1B knockdown with 3 independent shRNAs; i) Proliferation following shRNA-mediated ARID1B knockdown measured by MTT assay; error bars show standard deviation of 3 measurements (technical replicates).

    Journal: Nature genetics

    Article Title: ARID1A loss impairs enhancer-mediated gene regulation and drives colon cancer in mice

    doi: 10.1038/ng.3744

    Figure Lengend Snippet: ARID1A loss causes defects in SWI/SNF targeting to chromatin a) Live cell morphology of HCT116 ARID1A WT, ARID1A +/− , and Arid1a −/− cells in culture; b) Proliferation measured by MTT assay, error bars show standard deviation of 3 measurements (technical replicates); c) Invasion measured by Matrigel-chamber based assay, error bars show standard deviation of 4 measurements (technical replicates); d) Protein levels of ARID1A, E-Cadherin, and β-actin; e) Protein levels of Vimentin and β-actin (Note: NIH 3T3 fibroblast cells included for positive control); f) Fold change (log 2 ) in SMARCA4 and SMARCC1 ChIP-Seq signals at SWI/SNF binding sites in Arid1a −/− cells relative to WT; g) Immunoprecipitation of SWI/SNF complexes using antibodies targeting ARID1A and ARID1B; h) Protein levels of ARID1B and β-actin following ARID1B knockdown with 3 independent shRNAs; i) Proliferation following shRNA-mediated ARID1B knockdown measured by MTT assay; error bars show standard deviation of 3 measurements (technical replicates).

    Article Snippet: The following antibodies were used for immunoprecipitation of 30ug solubilized chromatin: SMARCC1/BAF155 (Santa Cruz sc9746; ChIP-Seq and ChIP-qPCR), SMARCA4/BRG1 (Abcam ab110641; ChIP-Seq and ChIP-qPCR), H3K27ac (Abcam ab4729; ChIP-Seq and ChIP-qPCR); H3K4me (Abcam ab8895; ChIP-Seq), H3K4me3 (Abcam ab8580; ChIP-Seq); ARID1A (Santa Cruz 32761 and Millipore PSG3; ChIP-qPCR) and ARID1B (Abcam ab57461 and Santa Cruz 32762; ChIP-qPCR).

    Techniques: MTT Assay, Standard Deviation, Boyden Chamber Assay, Positive Control, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, shRNA

    SWI/SNF complexes are targeted to enhancers and contribute to their activity a) Distribution of SWI/SNF binding sites in HCT116 WT and Arid1a −/− cells relative to histone modifications (Note: difference between WT and Arid1a −/− cells is not significant in a paired t-test); b) ChIP-seq profiles of SMARCA4, SMARCC1, H3K4me3, H3K4me1, and H3K27ac in WT and ARID1A −/− cells around all TSS-distal SWI/SNF binding sites (Notes: labels on the right of the figure indicate number of sites in each category; labels on top right corners indicate any alterations made in scaling of Y-axis); c) H3K27ac levels in WT and ARID1A −/− cells at TSS-proximal (promoter) and TSS-distal (enhancer) enrichment regions (Note: numbers in the three corners denote numbers of activated ( > 2×), inactivated (

    Journal: Nature genetics

    Article Title: ARID1A loss impairs enhancer-mediated gene regulation and drives colon cancer in mice

    doi: 10.1038/ng.3744

    Figure Lengend Snippet: SWI/SNF complexes are targeted to enhancers and contribute to their activity a) Distribution of SWI/SNF binding sites in HCT116 WT and Arid1a −/− cells relative to histone modifications (Note: difference between WT and Arid1a −/− cells is not significant in a paired t-test); b) ChIP-seq profiles of SMARCA4, SMARCC1, H3K4me3, H3K4me1, and H3K27ac in WT and ARID1A −/− cells around all TSS-distal SWI/SNF binding sites (Notes: labels on the right of the figure indicate number of sites in each category; labels on top right corners indicate any alterations made in scaling of Y-axis); c) H3K27ac levels in WT and ARID1A −/− cells at TSS-proximal (promoter) and TSS-distal (enhancer) enrichment regions (Note: numbers in the three corners denote numbers of activated ( > 2×), inactivated (

    Article Snippet: The following antibodies were used for immunoprecipitation of 30ug solubilized chromatin: SMARCC1/BAF155 (Santa Cruz sc9746; ChIP-Seq and ChIP-qPCR), SMARCA4/BRG1 (Abcam ab110641; ChIP-Seq and ChIP-qPCR), H3K27ac (Abcam ab4729; ChIP-Seq and ChIP-qPCR); H3K4me (Abcam ab8895; ChIP-Seq), H3K4me3 (Abcam ab8580; ChIP-Seq); ARID1A (Santa Cruz 32761 and Millipore PSG3; ChIP-qPCR) and ARID1B (Abcam ab57461 and Santa Cruz 32762; ChIP-qPCR).

    Techniques: Activity Assay, Binding Assay, Chromatin Immunoprecipitation

    Schematic of SMARCA4 mutations in SCCOHT. SMARCA4 mutations identified in germline and tumor DNA from 62 SCCOHT patients, and in 2 SCCOHT cell lines (Case 103 from Jelinic et al. with exon deletion is not shown). 1–4 QLQ, Gln, Leu, Gln motif; HSA, helicase/SANT-associated domain; BRK, brahma and kismet domain; DEXDc, DEAD-like helicase superfamily domain; HELICc, helicase superfamily C-terminal domain; Bromo, bromodomain.

    Journal: Rare Diseases

    Article Title: Loss of the tumor suppressor SMARCA4 in small cell carcinoma of the ovary, hypercalcemic type (SCCOHT)

    doi: 10.4161/2167549X.2014.967148

    Figure Lengend Snippet: Schematic of SMARCA4 mutations in SCCOHT. SMARCA4 mutations identified in germline and tumor DNA from 62 SCCOHT patients, and in 2 SCCOHT cell lines (Case 103 from Jelinic et al. with exon deletion is not shown). 1–4 QLQ, Gln, Leu, Gln motif; HSA, helicase/SANT-associated domain; BRK, brahma and kismet domain; DEXDc, DEAD-like helicase superfamily domain; HELICc, helicase superfamily C-terminal domain; Bromo, bromodomain.

    Article Snippet: Unstained slides were processed using the Ventana Discovery Ultra system (Ventana Medical Systems), using a rabbit monoclonal antibody to SMARCA4 (BRG1; Abcam, ab110641; 1:25 dilution) and mouse monoclonal antibody to SMARCB1 (INI1; BD Transduction Laboratories, 612110; 1:50 dilution).

    Techniques:

    Effect of SWI/SNF subunits BAF47, BAF53a and BRG1 knockdown on the irreversible cell cycle exit. C2C12 myoblasts were transfected with control, BRG1, BAF53a or BAF47 siRNAs and cells were labeled with BrdU either after 96 h in DM or 12 h after the switch back to GM (96+12 h GM). We show one representative of 3 independent experiments. Left panel: BrdU indexes ratio of BrdU positive nuclei/DAPI labeled nuclei) are shown in the histogram, error bars represent SEM from 3 quantifications of the presented experiment (*p

    Journal: PLoS ONE

    Article Title: The SWI/SNF Subunit/Tumor Suppressor BAF47/INI1 Is Essential in Cell Cycle Arrest upon Skeletal Muscle Terminal Differentiation

    doi: 10.1371/journal.pone.0108858

    Figure Lengend Snippet: Effect of SWI/SNF subunits BAF47, BAF53a and BRG1 knockdown on the irreversible cell cycle exit. C2C12 myoblasts were transfected with control, BRG1, BAF53a or BAF47 siRNAs and cells were labeled with BrdU either after 96 h in DM or 12 h after the switch back to GM (96+12 h GM). We show one representative of 3 independent experiments. Left panel: BrdU indexes ratio of BrdU positive nuclei/DAPI labeled nuclei) are shown in the histogram, error bars represent SEM from 3 quantifications of the presented experiment (*p

    Article Snippet: For ChIP experiments, we used anti-BRG1 (ab110641) and anti-BAF47 (ab12167) from Abcam.

    Techniques: Transfection, Labeling

    SWI/SNF complex subunits are differently involved in skeletal muscle terminal differentiation. A. C2C12 myoblasts were transfected with control, BRG1, BAF53a or BAF47 siRNAs. Immunofluorescence analyses using anti-MCK antibody were performed after 72h in DM. Cells were DAPI-stained prior to fluorescent microscopy analyses (magnification x20). B and C. C2C12 myoblasts were transfected with control, BRG1, BAF53a or BAF47 siRNAs. 24 h post-transfection (0) cells were placed in DM and harvested after 24 h, 48 h or 72 h. B. Total protein extracts were analyzed by WB with the indicated antibodies. *: non-specific bands. For each time point, % of protein siRNA-induced downregulation compared to the same time from control scrambled siRNA is indicated at the bottom of the western blot (see Figure S2 for additional WB analyses). C. Quantification of MCK levels from 3 to 8 independent western blot. Error bars represent standard error of the mean (SEM). For each time point, statistics were calculated compared to the same time point from control. Significative p-values (Student T-test, two tailed, unpaired) are indicated * =

    Journal: PLoS ONE

    Article Title: The SWI/SNF Subunit/Tumor Suppressor BAF47/INI1 Is Essential in Cell Cycle Arrest upon Skeletal Muscle Terminal Differentiation

    doi: 10.1371/journal.pone.0108858

    Figure Lengend Snippet: SWI/SNF complex subunits are differently involved in skeletal muscle terminal differentiation. A. C2C12 myoblasts were transfected with control, BRG1, BAF53a or BAF47 siRNAs. Immunofluorescence analyses using anti-MCK antibody were performed after 72h in DM. Cells were DAPI-stained prior to fluorescent microscopy analyses (magnification x20). B and C. C2C12 myoblasts were transfected with control, BRG1, BAF53a or BAF47 siRNAs. 24 h post-transfection (0) cells were placed in DM and harvested after 24 h, 48 h or 72 h. B. Total protein extracts were analyzed by WB with the indicated antibodies. *: non-specific bands. For each time point, % of protein siRNA-induced downregulation compared to the same time from control scrambled siRNA is indicated at the bottom of the western blot (see Figure S2 for additional WB analyses). C. Quantification of MCK levels from 3 to 8 independent western blot. Error bars represent standard error of the mean (SEM). For each time point, statistics were calculated compared to the same time point from control. Significative p-values (Student T-test, two tailed, unpaired) are indicated * =

    Article Snippet: For ChIP experiments, we used anti-BRG1 (ab110641) and anti-BAF47 (ab12167) from Abcam.

    Techniques: Transfection, Immunofluorescence, Staining, Microscopy, Western Blot, Two Tailed Test

    Downregulation of BAF47 alters muscle terminal differentiation and cell cycle exit. A. BAF47 and BRG1 occupancy at cyclin D1 promoter. ChIP-qPCR analyses of BAF47 and BRG1 in myoblast in proliferating C2C12 cells and at 24 h of differentiation. The immunoprecipitated material was quantified by qPCR, and results are expressed as fold enrichment of the % of Input of BAF47 or BRG1 ChIP over % of Input of the IgG average. Data are represented as mean ±SEM, n = 3. B. Scheme of the timing for samples collection. C2C12 myoblasts were transfected with control, BRG1, BAF53a or BAF47 siRNAs and samples prepared either 24 h after transfection (0) or after 72 h in DM (72) or 10 h after the switch back to GM (72+10 h GM). C. Total protein extracts were analyzed by WB with the indicated antibodies. D. Quantification of cyD1 levels from 3 to 8 independent WB. Data are expressed compared to control 0 h. Error bars represent SEM. For each time point, statistics were calculated compare to the same time point from control. Only significative p-values (Student T-test, two tailed, unpaired) are indicated ** =

    Journal: PLoS ONE

    Article Title: The SWI/SNF Subunit/Tumor Suppressor BAF47/INI1 Is Essential in Cell Cycle Arrest upon Skeletal Muscle Terminal Differentiation

    doi: 10.1371/journal.pone.0108858

    Figure Lengend Snippet: Downregulation of BAF47 alters muscle terminal differentiation and cell cycle exit. A. BAF47 and BRG1 occupancy at cyclin D1 promoter. ChIP-qPCR analyses of BAF47 and BRG1 in myoblast in proliferating C2C12 cells and at 24 h of differentiation. The immunoprecipitated material was quantified by qPCR, and results are expressed as fold enrichment of the % of Input of BAF47 or BRG1 ChIP over % of Input of the IgG average. Data are represented as mean ±SEM, n = 3. B. Scheme of the timing for samples collection. C2C12 myoblasts were transfected with control, BRG1, BAF53a or BAF47 siRNAs and samples prepared either 24 h after transfection (0) or after 72 h in DM (72) or 10 h after the switch back to GM (72+10 h GM). C. Total protein extracts were analyzed by WB with the indicated antibodies. D. Quantification of cyD1 levels from 3 to 8 independent WB. Data are expressed compared to control 0 h. Error bars represent SEM. For each time point, statistics were calculated compare to the same time point from control. Only significative p-values (Student T-test, two tailed, unpaired) are indicated ** =

    Article Snippet: For ChIP experiments, we used anti-BRG1 (ab110641) and anti-BAF47 (ab12167) from Abcam.

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Transfection, Western Blot, Two Tailed Test

    BRG1 and BAF47 interact with SWI/SNF and N-CoR-1 complex components in a differentiation-dependent manner. A. Nuclear extracts from proliferating (GM) or differentiating C2C12 myoblasts (24 h DM) were used for immunoprecipitation (IP) with antibodies against BRG1 or BAF47, or with normal rabbit IgG as a negative control. The resulting precipitates were analyzed by WB with the indicated antibodies. Nuclear extracts (Inputs, 1% of input extracts) were loaded to assess endogenous protein levels. *: non-specific IgG band. B. Nuclear extracts from proliferating (GM) or differentiating C2C12 myoblasts (24 h DM) were fractionated on glycerol gradient ranging from 11% (fraction 1) to 33% (fraction 11); (-) empty lane. Fractions were collected and analyzed by WB using the indicated antibodies. Nuclear extracts (Inputs, 2.5% of input extracts) were loaded to assess endogenous protein levels.

    Journal: PLoS ONE

    Article Title: The SWI/SNF Subunit/Tumor Suppressor BAF47/INI1 Is Essential in Cell Cycle Arrest upon Skeletal Muscle Terminal Differentiation

    doi: 10.1371/journal.pone.0108858

    Figure Lengend Snippet: BRG1 and BAF47 interact with SWI/SNF and N-CoR-1 complex components in a differentiation-dependent manner. A. Nuclear extracts from proliferating (GM) or differentiating C2C12 myoblasts (24 h DM) were used for immunoprecipitation (IP) with antibodies against BRG1 or BAF47, or with normal rabbit IgG as a negative control. The resulting precipitates were analyzed by WB with the indicated antibodies. Nuclear extracts (Inputs, 1% of input extracts) were loaded to assess endogenous protein levels. *: non-specific IgG band. B. Nuclear extracts from proliferating (GM) or differentiating C2C12 myoblasts (24 h DM) were fractionated on glycerol gradient ranging from 11% (fraction 1) to 33% (fraction 11); (-) empty lane. Fractions were collected and analyzed by WB using the indicated antibodies. Nuclear extracts (Inputs, 2.5% of input extracts) were loaded to assess endogenous protein levels.

    Article Snippet: For ChIP experiments, we used anti-BRG1 (ab110641) and anti-BAF47 (ab12167) from Abcam.

    Techniques: Immunoprecipitation, Negative Control, Western Blot

    BRG1 and STAT3 are recruited to STAT3 binding sites (SBS) at Gfap and Osmr loci concurrent with gene clustering. (A) Expression of BRG1 in GFAP-positive cells. NPCs alone and stimulated with LIF for different periods of time (LIF-stim) were stained with an anti-GFAP antibody (green) and an anti-BRG1 antibody (red). Nuclei were counterstained with DAPI (blue). Scale bar = 20 µm. Arrows indicate BRG1/GFAP double-positive cells. (B) The number of BRG1-positive cells in total cells. (C) The number of GFAP-positive and GFAP/BRG1–double positive cells. (B, C) Data are presented as the means ± SEM from three biological replicates ( n = 72–211). The Dunnett test was performed. ** p

    Journal: Molecular Biology of the Cell

    Article Title: Gfap and Osmr regulation by BRG1 and STAT3 via interchromosomal gene clustering in astrocytes

    doi: 10.1091/mbc.E17-05-0271

    Figure Lengend Snippet: BRG1 and STAT3 are recruited to STAT3 binding sites (SBS) at Gfap and Osmr loci concurrent with gene clustering. (A) Expression of BRG1 in GFAP-positive cells. NPCs alone and stimulated with LIF for different periods of time (LIF-stim) were stained with an anti-GFAP antibody (green) and an anti-BRG1 antibody (red). Nuclei were counterstained with DAPI (blue). Scale bar = 20 µm. Arrows indicate BRG1/GFAP double-positive cells. (B) The number of BRG1-positive cells in total cells. (C) The number of GFAP-positive and GFAP/BRG1–double positive cells. (B, C) Data are presented as the means ± SEM from three biological replicates ( n = 72–211). The Dunnett test was performed. ** p

    Article Snippet: A mouse monoclonal antibody (mAb) against GFAP (Sigma, #G-6171; 1:400), a rabbit mAb against BRG1 (Abcam, #ab110641; 1:400), and a chicken polyclonal antibody against GFP (Aves Lab, #GFP-1020; 1:200) were used as primary antibodies.

    Techniques: Binding Assay, Expressing, Staining

    BRG1 knockdown down-regulates Gfap and Osmr transcription and impairs gene clustering. (A) Scheme of cell culture and virus infection procedures. NPCs isolated from E14.5 mouse telencephalon were cultured and replated on day 4. Subsequently, the cells were infected with retroviruses that expressed EGFP alone (shControl) or EGFP together with shBRG1 and then cultured with LIF (50 ng/ml) for astrocyte differentiation. (B) Quantitative RT-PCR for Gfap mRNA in shControl- or shBRG1-treated NPCs alone or stimulated with LIF for different periods of time (LIF-stim). The results were normalized to Gapdh expression and described as fold induction relative to the levels at 0 h. Each graph represents the mean (±SEM) for the relative amount of NPCs in at least three experiments. (C) Projected images of double-labeled DNA FISH in GFP-positive (green) LIF-stimulated cells for Gfap (yellow) and Osmr (red). Nuclei were counterstained with DAPI (blue). Scale bar = 5 µm. Arrowheads indicate clustering alleles. (D) Clustering frequencies determined for Gfap and Osmr in LIF-stimulated cells expressing shControl or shBRG1. Data are presented as the means ± SEM from three biological replicates ( n = 73–127). Student’s t test was performed. ** p

    Journal: Molecular Biology of the Cell

    Article Title: Gfap and Osmr regulation by BRG1 and STAT3 via interchromosomal gene clustering in astrocytes

    doi: 10.1091/mbc.E17-05-0271

    Figure Lengend Snippet: BRG1 knockdown down-regulates Gfap and Osmr transcription and impairs gene clustering. (A) Scheme of cell culture and virus infection procedures. NPCs isolated from E14.5 mouse telencephalon were cultured and replated on day 4. Subsequently, the cells were infected with retroviruses that expressed EGFP alone (shControl) or EGFP together with shBRG1 and then cultured with LIF (50 ng/ml) for astrocyte differentiation. (B) Quantitative RT-PCR for Gfap mRNA in shControl- or shBRG1-treated NPCs alone or stimulated with LIF for different periods of time (LIF-stim). The results were normalized to Gapdh expression and described as fold induction relative to the levels at 0 h. Each graph represents the mean (±SEM) for the relative amount of NPCs in at least three experiments. (C) Projected images of double-labeled DNA FISH in GFP-positive (green) LIF-stimulated cells for Gfap (yellow) and Osmr (red). Nuclei were counterstained with DAPI (blue). Scale bar = 5 µm. Arrowheads indicate clustering alleles. (D) Clustering frequencies determined for Gfap and Osmr in LIF-stimulated cells expressing shControl or shBRG1. Data are presented as the means ± SEM from three biological replicates ( n = 73–127). Student’s t test was performed. ** p

    Article Snippet: A mouse monoclonal antibody (mAb) against GFAP (Sigma, #G-6171; 1:400), a rabbit mAb against BRG1 (Abcam, #ab110641; 1:400), and a chicken polyclonal antibody against GFP (Aves Lab, #GFP-1020; 1:200) were used as primary antibodies.

    Techniques: Cell Culture, Infection, Isolation, Quantitative RT-PCR, Expressing, Labeling, Fluorescence In Situ Hybridization

    JAK-STAT signaling is required for BRG1 recruitment to STAT3 binding sites (SBS) at Gfap and Osmr loci . (A) ChIP- and reChIP-qPCR for STAT3 and BRG1 at the SBS of Gfap in NPCs and cells stimulated with LIF for different periods of time (LIF-stim). Each graph represents the means (±SEM) of at least three experiments. (B) ChIP-qPCR for BRG1 on the SBS of Gfap, Osmr , and Gapdh (negative control) in cells stimulated with LIF for 48 h (LIF-stim 48 h). Each graph represents the means (±SEM) of at least three experiments. (C) Model depicting BRG1 as a mediator of gene clustering of Gfap and Osmr . BRG1 and STAT3 are recruited to the SBS of Gfap and Osmr within 48 h after LIF stimulation concurrent with BRG1- and STAT3-dependent Gfap and Osmr gene clustering. Transcription of both genes is enhanced after gene clustering (at 72–96 h after LIF stimulation).

    Journal: Molecular Biology of the Cell

    Article Title: Gfap and Osmr regulation by BRG1 and STAT3 via interchromosomal gene clustering in astrocytes

    doi: 10.1091/mbc.E17-05-0271

    Figure Lengend Snippet: JAK-STAT signaling is required for BRG1 recruitment to STAT3 binding sites (SBS) at Gfap and Osmr loci . (A) ChIP- and reChIP-qPCR for STAT3 and BRG1 at the SBS of Gfap in NPCs and cells stimulated with LIF for different periods of time (LIF-stim). Each graph represents the means (±SEM) of at least three experiments. (B) ChIP-qPCR for BRG1 on the SBS of Gfap, Osmr , and Gapdh (negative control) in cells stimulated with LIF for 48 h (LIF-stim 48 h). Each graph represents the means (±SEM) of at least three experiments. (C) Model depicting BRG1 as a mediator of gene clustering of Gfap and Osmr . BRG1 and STAT3 are recruited to the SBS of Gfap and Osmr within 48 h after LIF stimulation concurrent with BRG1- and STAT3-dependent Gfap and Osmr gene clustering. Transcription of both genes is enhanced after gene clustering (at 72–96 h after LIF stimulation).

    Article Snippet: A mouse monoclonal antibody (mAb) against GFAP (Sigma, #G-6171; 1:400), a rabbit mAb against BRG1 (Abcam, #ab110641; 1:400), and a chicken polyclonal antibody against GFP (Aves Lab, #GFP-1020; 1:200) were used as primary antibodies.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control

    Foxa2/H2A.Z-driven nucleosome depletion complexes during ES cell differentiation. ( A–C ) Smarca4 (Brg1), Kat5 (Tip60), and Nap1l1, are enriched at nucleosome depletion regions. The tag density was normalized to 10 million sequencing tags for each

    Journal: Cell

    Article Title: Foxa2 and H2A.Z Mediate Nucleosome Depletion during Embryonic Stem Cell Differentiation

    doi: 10.1016/j.cell.2012.11.018

    Figure Lengend Snippet: Foxa2/H2A.Z-driven nucleosome depletion complexes during ES cell differentiation. ( A–C ) Smarca4 (Brg1), Kat5 (Tip60), and Nap1l1, are enriched at nucleosome depletion regions. The tag density was normalized to 10 million sequencing tags for each

    Article Snippet: Antibodies used were: Foxa2 (a kind gift of J. Whitsett, Cincinnati), H2A.Z (Abcam, ab4174), H2A.X (Abcam, ab11175), Brg1 (Santa Cruz, sc-8749 and Abcam, ab4081), Tip60 (Santa Cruz, sc-5725 and Abcam, ab23886), and Nap1l1 (Santa Cruz, sc-292698 and Abcam, ab33076).

    Techniques: Cell Differentiation, Sequencing