anti-brg1 antibody Search Results


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  • 93
    Thermo Fisher brg1
    <t>Brg1</t> and Brm knockdown by shRNA. A , MLE-15 cells were transduced with retroviral vectors expressing a GFP reporter gene along with either a non-silencing control shRNA ( Control ) or shRNA targeting an mRNA sequence common to Brg1 and Brm coding regions
    Brg1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 99 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti brg1
    <t>Brg1,</t> Snf2h, and Chd4 tend to co-occupy the same genomic regions (a) Venn diagrams displaying overlaps of binding site occupancy between pairs of remodelers. (b) ChIP-seq genome browser view of Brg1 (blue track), Chd4 (green track), and Snf2h (dark red track) occupancy at the same genomic coordinates on chromosome 6. Mapped sequence tags represented as tag density are indicated on the y-axis. (c) An expanded view of the selected region in panel [(b)] . Displayed on the right-side is a three-way Venn diagram demonstrating the overlap between the binding sites of Brg1(blue), Chd4 (dark yellow), and Snf2h (red). (d) Distribution at annotated genic regions of shared and unique remodeler binding sites. Promoter represents region ± 2.5 kb from TSS.
    Anti Brg1, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti smarca4
    <t>SMARCA4/2</t> regulate CCND1 via controlling chromatin accessibility and upregulating JUN . a Assay for transposase-accessible chromatin sequencing (ATAC-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) data in vicinity of the CCND1 locus indicate enhanced chromatin accessibility upon SMARCA4/2 restoration. Note SMARCA4 at CCND1 promoter and formation of new putative enhancer ~50 kb upstream of CCND1 . Track height is normalized to relative number of mapped reads. b Zoomed-in view of the putative CCND1 enhancer region. Shown are ATAC-seq peaks in H1703 cells before and after SMARCA4/2 restoration and the publicly available c-Fos/c-Jun ChIP data of endothelial cell line, human umbilical vein endothelial cell (HUVEC) (GSM935585, GSM935278). Location of canonical adaptor protein-1 (AP-1) motifs are indicated. c ATAC and ChIP-seq data in vicinity of JUN locus as described in a . Note SMARCA4 at JUN promoter and extensive opening of nearby putative enhancers. d– i Restoration of SMARCA4 in H1703 ( d , e ) and H1299 ( f , g ) or SMARCA2 restoration in H1703 ( h , i ) cells upregulate c-Jun messenger RNA (mRNA) ( d , f , h ) and protein ( e , g , i ). j , k Knockdown of JUN partially abrogated SMARCA4-mediated induction of cyclin D1 mRNA ( j ) and protein ( k ) expression in H1703 cells. l Proposed model showing that SMARCA4 directly regulates CCND1 and also upregulates JUN which positively regulates CCND1 . Two-tailed t -test. Error bars represent mean ± s.d., *** p
    Anti Smarca4, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti brg1 antibody epncir111a
    <t>SMARCA4/2</t> regulate CCND1 via controlling chromatin accessibility and upregulating JUN . a Assay for transposase-accessible chromatin sequencing (ATAC-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) data in vicinity of the CCND1 locus indicate enhanced chromatin accessibility upon SMARCA4/2 restoration. Note SMARCA4 at CCND1 promoter and formation of new putative enhancer ~50 kb upstream of CCND1 . Track height is normalized to relative number of mapped reads. b Zoomed-in view of the putative CCND1 enhancer region. Shown are ATAC-seq peaks in H1703 cells before and after SMARCA4/2 restoration and the publicly available c-Fos/c-Jun ChIP data of endothelial cell line, human umbilical vein endothelial cell (HUVEC) (GSM935585, GSM935278). Location of canonical adaptor protein-1 (AP-1) motifs are indicated. c ATAC and ChIP-seq data in vicinity of JUN locus as described in a . Note SMARCA4 at JUN promoter and extensive opening of nearby putative enhancers. d– i Restoration of SMARCA4 in H1703 ( d , e ) and H1299 ( f , g ) or SMARCA2 restoration in H1703 ( h , i ) cells upregulate c-Jun messenger RNA (mRNA) ( d , f , h ) and protein ( e , g , i ). j , k Knockdown of JUN partially abrogated SMARCA4-mediated induction of cyclin D1 mRNA ( j ) and protein ( k ) expression in H1703 cells. l Proposed model showing that SMARCA4 directly regulates CCND1 and also upregulates JUN which positively regulates CCND1 . Two-tailed t -test. Error bars represent mean ± s.d., *** p
    Anti Brg1 Antibody Epncir111a, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 99 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology brg1 antibodies
    Nucleosome eviction does not correlate with chromatin modification, <t>BRG1</t> recruitment or H2A.Z deposition. The levels of H4Ac, H3Ac, H3K4Me3, BRG1 recruitment and H2A.Z deposition at the HLA-DRA promoter were analyzed by qChIP in wild-type Raji B cells (WT), CIITA-deficient B cells (CIITA −/− ) and RFX-deficient B cells (RFX −/− ). Results were corrected for nucleosome density using antibodies directed against unmodified histone H3, and were normalized relative to a control position at the TBP promoter. Results are expressed relative to the values observed in wild-type cells, and represent the means and standard deviations derived from three independent experiments. Primers used are indicated in Supplementary Table 1 .
    Brg1 Antibodies, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bethyl rabbit anti brg1 smarca4 antibody affinity purified
    <t>SMARCA4/2</t> regulate CCND1 via controlling chromatin accessibility and upregulating JUN . a Assay for transposase-accessible chromatin sequencing (ATAC-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) data in vicinity of the CCND1 locus indicate enhanced chromatin accessibility upon SMARCA4/2 restoration. Note SMARCA4 at CCND1 promoter and formation of new putative enhancer ~50 kb upstream of CCND1 promoter. All data were generated in H1703 cells before and after restoration of SMARCA4 or SMARCA2, except the publicly available SMARCA4 ChIP data in H1299 cells expressing doxycycline (Dox)-inducible SMARCA4 34 . Track height is normalized to relative number of mapped reads. b Zoomed-in view of the putative CCND1 enhancer region. Shown are ATAC-seq peaks in H1703 cells before and after SMARCA4/2 restoration and the publicly available c-Fos/c-Jun ChIP data of endothelial cell line, human umbilical vein endothelial cell (HUVEC) (GSM935585, GSM935278). Location of canonical adaptor protein-1 (AP-1) motifs are indicated. c ATAC and ChIP-seq data in vicinity of JUN locus as described in a . Note SMARCA4 at JUN promoter and extensive opening of nearby putative enhancers. d– i Restoration of SMARCA4 in H1703 ( d , e ) and H1299 ( f , g ) or SMARCA2 restoration in H1703 ( h , i ) cells upregulate c-Jun messenger RNA (mRNA) ( d , f , h ) and protein ( e , g , i ). j , k Knockdown of JUN partially abrogated SMARCA4-mediated induction of cyclin D1 mRNA ( j ) and protein ( k ) expression in H1703 cells. l Proposed model showing that SMARCA4 directly regulates CCND1 and also upregulates JUN which positively regulates CCND1 . Two-tailed t -test. Error bars represent mean ± s.d., *** p
    Rabbit Anti Brg1 Smarca4 Antibody Affinity Purified, supplied by Bethyl, used in various techniques. Bioz Stars score: 94/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology rabbit anti brg1 antibody
    <t>BRG1</t> does not alter p16 INK4a cell cycle regulation . A 50 μg of nuclear lysates from WMM1175_p16 INK4a clones with stably integrated siRNA targeting BRG1 or a non-specific (NS) control siRNA were probed for BRG1 and topoisomerase II (Topo II) as a loading control. B 50 μg of total cell lysates extracted from WMM1175_p16 INK4a cells stably expressing either a BRG1-specific siRNA or a non-specific (NS) siRNA molecule, as indicated, were treated with PBS (-) or IPTG (+) for 24 h and probed for p16 INK4a and β-actin. C Cell proliferation was determined by MTS assay. D A proportion of the IPTG/mock treated cells were analyzed for changes in cell cycle distribution. Percent S-phase change was calculated (percent S-phase mock treated cells – percent S-phase IPTG treated cells) × 100/percent S-phase mock treated cells. E The same clones were seeded at low density (10 3 cells/7.5 cm plate) and p16 INK4a expression was induced with 4 mM IPTG or cells mock treated and colony forming ability was assayed after 14 days.
    Rabbit Anti Brg1 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti brg1 antibody
    Telomerase reverse transcriptase (TERT) serves as a transcriptional modulator to regulate FASL expression in Bone marrow mesenchymal stem cells (BMMSCs). A–B Western blot analysis showed decreased levels of FASL and active β-catenin, but not <t>BRG1,</t> in TERT − / − BMMSCs (A) and tert knockdown BMMSCs by siRNA (B) compared to TERT +/+ (WT) BMMSCs. C β-catenin activator (Chir, 10 μM) treatment elevated levels of active β-catenin and FASL in WT BMMSCs. fasl knockdown BMMSCs by siRNA showed a decreased level of FASL expression, but not active β-catenin. D In vitro coculture system showed β-catenin activator (Chir)-treated BMMSCs had increased capacity to induce AnnexinV + 7AAD − and AnnexinV + 7AAD + double positive apoptotic T cells compared to control group. fasl siRNA treatment could reduce Chir-elevated T cell apoptosis in the co-culture system. E Telomerase activity in Chir-treated BMMSCs showed no significant difference from the untreated group. 293T cells were used as a positive control, and heat-inactivated (H.I.) samples were used as a negative control. F Western blot analysis showed decreased expression levels of β-catenin and FASL in β-catenin knockdown BMMSCs by siRNA. G β-catenin knockdown BMMSCs by siRNA showed decreased capacity to induce AnnexinV + 7AAD − and AnnexinV + 7AAD + double positive apoptotic T cells compared to the control siRNA group. H Western blot showed that TERT − / − BMMSCs decreased expression levels of TERT, active β-catenin, and FASL. Tert transfection (TERT TF) rescued the expression levels of TERT, active β-catenin, and FASL, assessed by Western blot, while fasl transfection (FASL TF) only rescued FASL expression, but not that of TERT or β-catenin, in TERT − / − BMMSCs. I In vitro coculture system showed a decreased capacity of TERT − / − BMMSCs to induce AnnexinV + 7AAD − and AnnexinV + 7AAD + double positive apoptotic T cells when compared to the control group, whereas transfection of both tert and fasl rescued the capacity to induce AnnexinV + 7AAD − and AnnexinV + 7AAD + double positive apoptotic T cells. J fasl promoter-luciferase fusions were examined in WT, TERT − / − and TERT TF BMMSCs. Promoter activity was expressed as relative light units (RLU) normalized to the activity of co-transfected Renilla luciferase. The activity of 2 kb promoter-luciferase fusion was significantly elevated compared to 1.1 kb fusion in WT BMMSCs and TERT TF BMMSCs when compared to TERT − / − BMMSCs. The activity of TBE-specific site-mutated promoters was markedly decreased in WT BMMSCs and TERT TF BMMSCs. K Chromatin immunoprecipitation (ChIP)-qPCR assay showed enrichment of direct association of β-catenin on the fasl promoter in WT and TERT − / − BMMSCs, while the enrichment of direct association of TERT on the fasl promoter was only found in WT BMMSCs. L ChIP-Western blot assays showed direct association of TERT, β-catenin and BRG1 on the fasl promoter in WT BMMSCs, but only direct association of β-catenin and BRG1 on the fasl promoter in TERT − / − BMMSCs. M Schematic diagram indicates that TERT, as a transcriptional modulator in a complex with β-catenin and BRG1, mediates FASL expression in BMMSC-induced immunoregulation. Vehicle: scrambled siRNA-treated BMMSCs. Data information: Error bars represent the s.d. from the mean values (One-way ANOVA, Bonferroni, n = 3 in each group, *** P
    Anti Brg1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 89/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam antibodies against brg1
    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
    Antibodies Against Brg1, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bethyl goat anti brg1 smarca4 antibody affinity purified
    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
    Goat Anti Brg1 Smarca4 Antibody Affinity Purified, supplied by Bethyl, used in various techniques. Bioz Stars score: 95/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti brg1 antibody epr3912
    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 Antibody Epr3912, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Molecular Biosciences Inc anti brg1 antibody
    β-actin is required for the regulation of chromatin status and the expression of Zic and Irx genes in MEFs or CiNeurons. (A) The relative expression of Zic amd Irx genes in MEFs and CiNeurons. Data are summary of at least 3 biological replicates from RNA-seq analysis. Error bar: S.E.M. (B) H3K9Me3 and <t>Brg1</t> ChIP-seq analysis in WTM and KOM cells at Zic and Irx loci. Normalized signal at Zic1 ; Zic2 ; Zic3 ; Zic4 ; Irx1 ; and Irx3 loci were shown. The y-axis data range represents RPKM (Reads Per Kilobase of sequence range per Million mapped reads) per bin. The y-axis of tracks in the same image were set as the same range. Gene body position (exon: box, intron: line) are shown below the tracks. Regions of each gene loci with elevated H3K9Me3 level and impaired Brg1 binding in KOM cells are highlighted.
    Anti Brg1 Antibody, supplied by Molecular Biosciences Inc, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti brg1 antibody
    <t>BRG1</t> protein expression in NSCs (a) Western immunoblot shows a single BRG1 band at ~238KD detected in the nuclear extract of neurosphere cultures but not in the cytoplasmic extract. (b) BRG1 protein levels in neurospheres after 0mg/dl, 120mg/dl and 320mg/dl ethanol treatment. Lower panel shows the corresponding immunoblot when probed for β-Actin as a loading control. (c) Bar graph, depicting the quantification of the western blot, shows that ethanol did not alter BRG1 protein expression, consistent with the lack of effect on BRG1 mRNA expression. The vertical axis shows the ratio of the band density of BRG1 to the ratio of the band density of β-Actin. Error bars indicates standard error of the mean. (d) Western blot analysis of BRG1 immunoprecipitation in cytoplasmic and nuclear cellular fractions probed with anti-BRG1 antibody. BRG1 is specifically precipitated with anti-BRG1 antibody (blue text), but not with an isotype-specific IgG control antibody (red text). Efficiency of anti-BRG1 immunoprecipitation is indicated by the relative depletion of BRG1 from the nuclear supernatant and enrichment in the immuno-precipitate (IP).
    Anti Brg1 Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti brg1 hsnf2beta antibody
    <t>BRG1</t> protein expression in NSCs (a) Western immunoblot shows a single BRG1 band at ~238KD detected in the nuclear extract of neurosphere cultures but not in the cytoplasmic extract. (b) BRG1 protein levels in neurospheres after 0mg/dl, 120mg/dl and 320mg/dl ethanol treatment. Lower panel shows the corresponding immunoblot when probed for β-Actin as a loading control. (c) Bar graph, depicting the quantification of the western blot, shows that ethanol did not alter BRG1 protein expression, consistent with the lack of effect on BRG1 mRNA expression. The vertical axis shows the ratio of the band density of BRG1 to the ratio of the band density of β-Actin. Error bars indicates standard error of the mean. (d) Western blot analysis of BRG1 immunoprecipitation in cytoplasmic and nuclear cellular fractions probed with anti-BRG1 antibody. BRG1 is specifically precipitated with anti-BRG1 antibody (blue text), but not with an isotype-specific IgG control antibody (red text). Efficiency of anti-BRG1 immunoprecipitation is indicated by the relative depletion of BRG1 from the nuclear supernatant and enrichment in the immuno-precipitate (IP).
    Anti Brg1 Hsnf2beta Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti brg1 sc 17796 antibody
    <t>BRG1</t> protein expression in NSCs (a) Western immunoblot shows a single BRG1 band at ~238KD detected in the nuclear extract of neurosphere cultures but not in the cytoplasmic extract. (b) BRG1 protein levels in neurospheres after 0mg/dl, 120mg/dl and 320mg/dl ethanol treatment. Lower panel shows the corresponding immunoblot when probed for β-Actin as a loading control. (c) Bar graph, depicting the quantification of the western blot, shows that ethanol did not alter BRG1 protein expression, consistent with the lack of effect on BRG1 mRNA expression. The vertical axis shows the ratio of the band density of BRG1 to the ratio of the band density of β-Actin. Error bars indicates standard error of the mean. (d) Western blot analysis of BRG1 immunoprecipitation in cytoplasmic and nuclear cellular fractions probed with anti-BRG1 antibody. BRG1 is specifically precipitated with anti-BRG1 antibody (blue text), but not with an isotype-specific IgG control antibody (red text). Efficiency of anti-BRG1 immunoprecipitation is indicated by the relative depletion of BRG1 from the nuclear supernatant and enrichment in the immuno-precipitate (IP).
    Anti Brg1 Sc 17796 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Brg1 and Brm knockdown by shRNA. A , MLE-15 cells were transduced with retroviral vectors expressing a GFP reporter gene along with either a non-silencing control shRNA ( Control ) or shRNA targeting an mRNA sequence common to Brg1 and Brm coding regions

    Journal:

    Article Title: Epigenetic Mechanisms Modulate Thyroid Transcription Factor 1-mediated Transcription of the Surfactant Protein B Gene *

    doi: 10.1074/jbc.M109.039172

    Figure Lengend Snippet: Brg1 and Brm knockdown by shRNA. A , MLE-15 cells were transduced with retroviral vectors expressing a GFP reporter gene along with either a non-silencing control shRNA ( Control ) or shRNA targeting an mRNA sequence common to Brg1 and Brm coding regions

    Article Snippet: For Brg1 and Brm mRNA analyses, we used SYBR Green Master Mix (Applied Biosystems).

    Techniques: shRNA, Transduction, Expressing, Sequencing

    Brg1 co-immunoprecipitates with Nkx2-1. A , MLE-15 cell lysates ( Input ) and immunoprecipitates ( IP ) obtained with rabbit IgG control, α-Brg1 antibody, or α -Nkx2-1 antibody were analyzed by Western blot with α-Brg1 antibody (immunoblot

    Journal:

    Article Title: Epigenetic Mechanisms Modulate Thyroid Transcription Factor 1-mediated Transcription of the Surfactant Protein B Gene *

    doi: 10.1074/jbc.M109.039172

    Figure Lengend Snippet: Brg1 co-immunoprecipitates with Nkx2-1. A , MLE-15 cell lysates ( Input ) and immunoprecipitates ( IP ) obtained with rabbit IgG control, α-Brg1 antibody, or α -Nkx2-1 antibody were analyzed by Western blot with α-Brg1 antibody (immunoblot

    Article Snippet: For Brg1 and Brm mRNA analyses, we used SYBR Green Master Mix (Applied Biosystems).

    Techniques: Western Blot

    Brg1 binds to the Sftpb gene promoter in mouse lung and MLE-15 cells that express Sftpb but not in mouse liver and E10 cells that do not express Sftpb. In these ChIP analyses DNA purified from immunoprecipitates with α-Brg1 antibody or its corresponding

    Journal:

    Article Title: Epigenetic Mechanisms Modulate Thyroid Transcription Factor 1-mediated Transcription of the Surfactant Protein B Gene *

    doi: 10.1074/jbc.M109.039172

    Figure Lengend Snippet: Brg1 binds to the Sftpb gene promoter in mouse lung and MLE-15 cells that express Sftpb but not in mouse liver and E10 cells that do not express Sftpb. In these ChIP analyses DNA purified from immunoprecipitates with α-Brg1 antibody or its corresponding

    Article Snippet: For Brg1 and Brm mRNA analyses, we used SYBR Green Master Mix (Applied Biosystems).

    Techniques: Chromatin Immunoprecipitation, Purification

    BRD4 recruits GBAF to target gene sites in a BD-dependent manner. a Scatterplot of the mRNA log2 FCs in I-BRD9/DMSO and JQ1/vehicle for 664 common DEGs. Linear regression analysis was performed to calculate the R 2 , with the best fit shown as a pink dashed line. b Venn diagram of overlaps between BRG1, BRD9, and BRD4 ChIP binding sites in ESCs, with n representing the number of observed peaks. c ESCs were treated with either DMSO or 200 nM of HDAC inhibitor trichostatin A (TSA) for 6 h prior to nuclear lysate collection then IP experiments were performed against BRD9 with or without I-BRD9. Molecular weights from ladder are indicated. d Quantification of BRD4 signal normalized to BRD9 bait signal then normalized to untreated from two biological experiments labeled a and b. Source data are provided as Source Data file. e Scatterplot of log2-transformed BRD4 ChIP tags in DMSO- and I-BRD9-treated ESCs. Blue and red correspond to 1.5-fold decrease or increase of BRD4 tag count in I-BRD9-treated ESCs, respectively (Poisson p value

    Journal: Nature Communications

    Article Title: A non-canonical BRD9-containing BAF chromatin remodeling complex regulates naive pluripotency in mouse embryonic stem cells

    doi: 10.1038/s41467-018-07528-9

    Figure Lengend Snippet: BRD4 recruits GBAF to target gene sites in a BD-dependent manner. a Scatterplot of the mRNA log2 FCs in I-BRD9/DMSO and JQ1/vehicle for 664 common DEGs. Linear regression analysis was performed to calculate the R 2 , with the best fit shown as a pink dashed line. b Venn diagram of overlaps between BRG1, BRD9, and BRD4 ChIP binding sites in ESCs, with n representing the number of observed peaks. c ESCs were treated with either DMSO or 200 nM of HDAC inhibitor trichostatin A (TSA) for 6 h prior to nuclear lysate collection then IP experiments were performed against BRD9 with or without I-BRD9. Molecular weights from ladder are indicated. d Quantification of BRD4 signal normalized to BRD9 bait signal then normalized to untreated from two biological experiments labeled a and b. Source data are provided as Source Data file. e Scatterplot of log2-transformed BRD4 ChIP tags in DMSO- and I-BRD9-treated ESCs. Blue and red correspond to 1.5-fold decrease or increase of BRD4 tag count in I-BRD9-treated ESCs, respectively (Poisson p value

    Article Snippet: After clarification of insoluble material by centrifugation, the chromatin was immunoprecipitated overnight at 4 °C with antibodies against BRG1, BRD9, and ARID1A bound to Protein A + G Dynabeads (Invitrogen) in ChIP buffer (50 mM HEPES-KOH, pH 7.5, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% DOC, and 0.1% SDS).

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Labeling, Transformation Assay

    GBAF and esBAF are co-bound by different pluripotency transcription factors. a Significance of enriched motifs for BRG1 sites uniquely bound by either ARID1A (top) or BRD9 (bottom). p Values were calculated using cumulative binomial distribution (findMotifsGenome.pl in HOMER). b Histogram of ChIP reads of the indicated transcription factor (TF) ± 1 kb surrounding the peak center of sites that are co-bound by either ARID1A and BRG1 or BRD9 and BRG1. c Representative genome browser tracks showing co-binding of BRD9 and BRG1 with either KLF4 or Sp5 at promoters (middle and right, respectively) or ARID1A and BRG1 with OCT4, SOX2, and NANOG at distal sites (left)

    Journal: Nature Communications

    Article Title: A non-canonical BRD9-containing BAF chromatin remodeling complex regulates naive pluripotency in mouse embryonic stem cells

    doi: 10.1038/s41467-018-07528-9

    Figure Lengend Snippet: GBAF and esBAF are co-bound by different pluripotency transcription factors. a Significance of enriched motifs for BRG1 sites uniquely bound by either ARID1A (top) or BRD9 (bottom). p Values were calculated using cumulative binomial distribution (findMotifsGenome.pl in HOMER). b Histogram of ChIP reads of the indicated transcription factor (TF) ± 1 kb surrounding the peak center of sites that are co-bound by either ARID1A and BRG1 or BRD9 and BRG1. c Representative genome browser tracks showing co-binding of BRD9 and BRG1 with either KLF4 or Sp5 at promoters (middle and right, respectively) or ARID1A and BRG1 with OCT4, SOX2, and NANOG at distal sites (left)

    Article Snippet: After clarification of insoluble material by centrifugation, the chromatin was immunoprecipitated overnight at 4 °C with antibodies against BRG1, BRD9, and ARID1A bound to Protein A + G Dynabeads (Invitrogen) in ChIP buffer (50 mM HEPES-KOH, pH 7.5, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% DOC, and 0.1% SDS).

    Techniques: Chromatin Immunoprecipitation, Binding Assay

    GBAF and esBAF localize to distinct genomic elements. a Venn diagram overlap of BRG1, BRD9, and ARID1A ChIP sites, with n representing the number of observed peaks. b Heat map of BRD9 and BRG1 ChIP signal ± 3 kilobases (kb) centered on the BRD9 peak, ranked according to BRD9 read density. c Significance of binding overlap between BRD9, BRG1, and ARID1A ChIP sites. The natural log of p values were calculated using hypergeometric distribution (mergePeaks.pl in HOMER). d Co-occurrence analysis showing the natural log of the ratio of the observed number of overlapping peaks over the expected values. This was done for BRD9, BRG1, and ARID1A ChIP sites against publicly available ChIP-Seq data for the histone modifications H3K4me, H3K4me3, H3K27me3, and H3K27ac. Poised enhancers are defined by sites that contain H3K4me1 but lack H3K27ac, whereas active enhancers are sites that are positive for both modifications. Super enhancers are H3K4me+, H3K27ac+, and Med1-high sites. e ChIP-Seq signal of BRG1, BRD9, and ARID1A ± 2 kb surrounding BRG1 peak center at promoters and distal sites. f ChIP-Seq signal of BRG1, BRD9, and ARID1A ± 50% size of a topologically associating domain (TAD) around a TAD

    Journal: Nature Communications

    Article Title: A non-canonical BRD9-containing BAF chromatin remodeling complex regulates naive pluripotency in mouse embryonic stem cells

    doi: 10.1038/s41467-018-07528-9

    Figure Lengend Snippet: GBAF and esBAF localize to distinct genomic elements. a Venn diagram overlap of BRG1, BRD9, and ARID1A ChIP sites, with n representing the number of observed peaks. b Heat map of BRD9 and BRG1 ChIP signal ± 3 kilobases (kb) centered on the BRD9 peak, ranked according to BRD9 read density. c Significance of binding overlap between BRD9, BRG1, and ARID1A ChIP sites. The natural log of p values were calculated using hypergeometric distribution (mergePeaks.pl in HOMER). d Co-occurrence analysis showing the natural log of the ratio of the observed number of overlapping peaks over the expected values. This was done for BRD9, BRG1, and ARID1A ChIP sites against publicly available ChIP-Seq data for the histone modifications H3K4me, H3K4me3, H3K27me3, and H3K27ac. Poised enhancers are defined by sites that contain H3K4me1 but lack H3K27ac, whereas active enhancers are sites that are positive for both modifications. Super enhancers are H3K4me+, H3K27ac+, and Med1-high sites. e ChIP-Seq signal of BRG1, BRD9, and ARID1A ± 2 kb surrounding BRG1 peak center at promoters and distal sites. f ChIP-Seq signal of BRG1, BRD9, and ARID1A ± 50% size of a topologically associating domain (TAD) around a TAD

    Article Snippet: After clarification of insoluble material by centrifugation, the chromatin was immunoprecipitated overnight at 4 °C with antibodies against BRG1, BRD9, and ARID1A bound to Protein A + G Dynabeads (Invitrogen) in ChIP buffer (50 mM HEPES-KOH, pH 7.5, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% DOC, and 0.1% SDS).

    Techniques: Chromatin Immunoprecipitation, Binding Assay

    Brg1, Snf2h, and Chd4 tend to co-occupy the same genomic regions (a) Venn diagrams displaying overlaps of binding site occupancy between pairs of remodelers. (b) ChIP-seq genome browser view of Brg1 (blue track), Chd4 (green track), and Snf2h (dark red track) occupancy at the same genomic coordinates on chromosome 6. Mapped sequence tags represented as tag density are indicated on the y-axis. (c) An expanded view of the selected region in panel [(b)] . Displayed on the right-side is a three-way Venn diagram demonstrating the overlap between the binding sites of Brg1(blue), Chd4 (dark yellow), and Snf2h (red). (d) Distribution at annotated genic regions of shared and unique remodeler binding sites. Promoter represents region ± 2.5 kb from TSS.

    Journal: Nature structural & molecular biology

    Article Title: Overlapping Chromatin Remodeling Systems Collaborate Genome-wide at Dynamic Chromatin Transitions

    doi: 10.1038/nsmb.2718

    Figure Lengend Snippet: Brg1, Snf2h, and Chd4 tend to co-occupy the same genomic regions (a) Venn diagrams displaying overlaps of binding site occupancy between pairs of remodelers. (b) ChIP-seq genome browser view of Brg1 (blue track), Chd4 (green track), and Snf2h (dark red track) occupancy at the same genomic coordinates on chromosome 6. Mapped sequence tags represented as tag density are indicated on the y-axis. (c) An expanded view of the selected region in panel [(b)] . Displayed on the right-side is a three-way Venn diagram demonstrating the overlap between the binding sites of Brg1(blue), Chd4 (dark yellow), and Snf2h (red). (d) Distribution at annotated genic regions of shared and unique remodeler binding sites. Promoter represents region ± 2.5 kb from TSS.

    Article Snippet: Blocked membranes were incubated overnight at 4°C with the following antibodies in TBST, 5% milk: anti-BRG1 (1:30,000), anti-CHD4 (1:2,000), anti-SNF2H (1:2,000), anti-WSTF (1:500, W3641, Sigma-Aldrich), anti-BAF155 (1:500, B5186, Sigma-Aldrich), and anti-HDAC1 (1:4,000, PA1-860, Pierce).

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Sequencing

    Remodeler binding sites are associated with DNA sequence-specific regulatory elements (a) Results of de novo motif discovery using the top 2,000 binding sites (based on tag density) co-occupied by Brg1, Chd4, and Snf2h. Shown are the most significantly enriched motifs identified by MEME analysis (P

    Journal: Nature structural & molecular biology

    Article Title: Overlapping Chromatin Remodeling Systems Collaborate Genome-wide at Dynamic Chromatin Transitions

    doi: 10.1038/nsmb.2718

    Figure Lengend Snippet: Remodeler binding sites are associated with DNA sequence-specific regulatory elements (a) Results of de novo motif discovery using the top 2,000 binding sites (based on tag density) co-occupied by Brg1, Chd4, and Snf2h. Shown are the most significantly enriched motifs identified by MEME analysis (P

    Article Snippet: Blocked membranes were incubated overnight at 4°C with the following antibodies in TBST, 5% milk: anti-BRG1 (1:30,000), anti-CHD4 (1:2,000), anti-SNF2H (1:2,000), anti-WSTF (1:500, W3641, Sigma-Aldrich), anti-BAF155 (1:500, B5186, Sigma-Aldrich), and anti-HDAC1 (1:4,000, PA1-860, Pierce).

    Techniques: Binding Assay, Sequencing

    Remodeler protein binding highly overlaps with accessible chromatin regions (a) Genome browser view examples of remodeler ChIP-seq occupancy and DNase I hypersensitivity (measure of chromatin accessibility, DNaseI-seq) patterns. Images represent tag densities (mapped sequence tags) relative to genome coordinates. Examples of binding sites that do not overlap with accessible chromatin are highlighted by grey shading. (b) Venn diagrams representing the overlap of binding sites for each remodeler with DNase I hypersensitive (DHS) sites. (c) Genome browser view of Brg1 (blue track), Chd4 (green track), and Snf2h (dark red track) ChIP-seq occupancy and DNaseI-seq patterns at a region on chromosome 6 are displayed. An expanded view of the selected region is shown below this image. Displayed on the right-side is a three-way Venn diagram representing the overlap between remodeler sites that specifically co-localize with DHS sites.

    Journal: Nature structural & molecular biology

    Article Title: Overlapping Chromatin Remodeling Systems Collaborate Genome-wide at Dynamic Chromatin Transitions

    doi: 10.1038/nsmb.2718

    Figure Lengend Snippet: Remodeler protein binding highly overlaps with accessible chromatin regions (a) Genome browser view examples of remodeler ChIP-seq occupancy and DNase I hypersensitivity (measure of chromatin accessibility, DNaseI-seq) patterns. Images represent tag densities (mapped sequence tags) relative to genome coordinates. Examples of binding sites that do not overlap with accessible chromatin are highlighted by grey shading. (b) Venn diagrams representing the overlap of binding sites for each remodeler with DNase I hypersensitive (DHS) sites. (c) Genome browser view of Brg1 (blue track), Chd4 (green track), and Snf2h (dark red track) ChIP-seq occupancy and DNaseI-seq patterns at a region on chromosome 6 are displayed. An expanded view of the selected region is shown below this image. Displayed on the right-side is a three-way Venn diagram representing the overlap between remodeler sites that specifically co-localize with DHS sites.

    Article Snippet: Blocked membranes were incubated overnight at 4°C with the following antibodies in TBST, 5% milk: anti-BRG1 (1:30,000), anti-CHD4 (1:2,000), anti-SNF2H (1:2,000), anti-WSTF (1:500, W3641, Sigma-Aldrich), anti-BAF155 (1:500, B5186, Sigma-Aldrich), and anti-HDAC1 (1:4,000, PA1-860, Pierce).

    Techniques: Protein Binding, Chromatin Immunoprecipitation, Sequencing, Binding Assay

    Remodeler proteins bind to distinct regions of chromatin (a) Example ChIP-seq genome browser views of Brg1 (top, blue tracks), Chd4 (middle, green tracks), and Snf2h (bottom, dark red tracks) occupancy. Images represent tag densities (mapped sequence tags) relative to genome coordinates. For each remodeler, the lower browser image displays an expanded view of the selected region where examples of localized distributions (single peak,

    Journal: Nature structural & molecular biology

    Article Title: Overlapping Chromatin Remodeling Systems Collaborate Genome-wide at Dynamic Chromatin Transitions

    doi: 10.1038/nsmb.2718

    Figure Lengend Snippet: Remodeler proteins bind to distinct regions of chromatin (a) Example ChIP-seq genome browser views of Brg1 (top, blue tracks), Chd4 (middle, green tracks), and Snf2h (bottom, dark red tracks) occupancy. Images represent tag densities (mapped sequence tags) relative to genome coordinates. For each remodeler, the lower browser image displays an expanded view of the selected region where examples of localized distributions (single peak,

    Article Snippet: Blocked membranes were incubated overnight at 4°C with the following antibodies in TBST, 5% milk: anti-BRG1 (1:30,000), anti-CHD4 (1:2,000), anti-SNF2H (1:2,000), anti-WSTF (1:500, W3641, Sigma-Aldrich), anti-BAF155 (1:500, B5186, Sigma-Aldrich), and anti-HDAC1 (1:4,000, PA1-860, Pierce).

    Techniques: Chromatin Immunoprecipitation, Sequencing

    SMARCA4/2 regulate CCND1 via controlling chromatin accessibility and upregulating JUN . a Assay for transposase-accessible chromatin sequencing (ATAC-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) data in vicinity of the CCND1 locus indicate enhanced chromatin accessibility upon SMARCA4/2 restoration. Note SMARCA4 at CCND1 promoter and formation of new putative enhancer ~50 kb upstream of CCND1 . Track height is normalized to relative number of mapped reads. b Zoomed-in view of the putative CCND1 enhancer region. Shown are ATAC-seq peaks in H1703 cells before and after SMARCA4/2 restoration and the publicly available c-Fos/c-Jun ChIP data of endothelial cell line, human umbilical vein endothelial cell (HUVEC) (GSM935585, GSM935278). Location of canonical adaptor protein-1 (AP-1) motifs are indicated. c ATAC and ChIP-seq data in vicinity of JUN locus as described in a . Note SMARCA4 at JUN promoter and extensive opening of nearby putative enhancers. d– i Restoration of SMARCA4 in H1703 ( d , e ) and H1299 ( f , g ) or SMARCA2 restoration in H1703 ( h , i ) cells upregulate c-Jun messenger RNA (mRNA) ( d , f , h ) and protein ( e , g , i ). j , k Knockdown of JUN partially abrogated SMARCA4-mediated induction of cyclin D1 mRNA ( j ) and protein ( k ) expression in H1703 cells. l Proposed model showing that SMARCA4 directly regulates CCND1 and also upregulates JUN which positively regulates CCND1 . Two-tailed t -test. Error bars represent mean ± s.d., *** p

    Journal: Nature Communications

    Article Title: SMARCA4 loss is synthetic lethal with CDK4/6 inhibition in non-small cell lung cancer

    doi: 10.1038/s41467-019-08380-1

    Figure Lengend Snippet: SMARCA4/2 regulate CCND1 via controlling chromatin accessibility and upregulating JUN . a Assay for transposase-accessible chromatin sequencing (ATAC-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) data in vicinity of the CCND1 locus indicate enhanced chromatin accessibility upon SMARCA4/2 restoration. Note SMARCA4 at CCND1 promoter and formation of new putative enhancer ~50 kb upstream of CCND1 . Track height is normalized to relative number of mapped reads. b Zoomed-in view of the putative CCND1 enhancer region. Shown are ATAC-seq peaks in H1703 cells before and after SMARCA4/2 restoration and the publicly available c-Fos/c-Jun ChIP data of endothelial cell line, human umbilical vein endothelial cell (HUVEC) (GSM935585, GSM935278). Location of canonical adaptor protein-1 (AP-1) motifs are indicated. c ATAC and ChIP-seq data in vicinity of JUN locus as described in a . Note SMARCA4 at JUN promoter and extensive opening of nearby putative enhancers. d– i Restoration of SMARCA4 in H1703 ( d , e ) and H1299 ( f , g ) or SMARCA2 restoration in H1703 ( h , i ) cells upregulate c-Jun messenger RNA (mRNA) ( d , f , h ) and protein ( e , g , i ). j , k Knockdown of JUN partially abrogated SMARCA4-mediated induction of cyclin D1 mRNA ( j ) and protein ( k ) expression in H1703 cells. l Proposed model showing that SMARCA4 directly regulates CCND1 and also upregulates JUN which positively regulates CCND1 . Two-tailed t -test. Error bars represent mean ± s.d., *** p

    Article Snippet: IHC was performed on the TMAs using anti-SMARCA4 (anti-BRG1 antibody (clone EPNCIR111A, dilution, 1:100 dilution, Abcam, Cambridge, UK), anti-p16 (clone G175-405, 1:20 dilution, BD PharmingenTM ) and anti-RB1 (G3-245, 1:100 dilution, BD PharmingenTM ) antibodies.

    Techniques: Sequencing, ChIP-sequencing, Chromatin Immunoprecipitation, Expressing, Two Tailed Test

    SMARCA4 loss is synthetic lethal with cyclin-dependent kinase 4/6 (CDK4/6) inhibition in non-small cell lung cancer (NSCLC). a , b SMARCA4 restoration in SMARCA4-deficient cell lines confers drug resistance to palbociclib. Colony formation assays of H1299 ( a ) and H1703 ( b ) cells expressing vector control or SMARCA4 and treated with palbociclib (H1299, 300 nM; H1703, 100 nM). c SMARCA2 knockdown in SMARCA4-deficient H1299 cells sensitizes cells to palbociclib treatment. Colony formation assay of H1299 cells expressing pLKO control or SMARCA2 short hairpin RNAs (shRNAs) and treated with palbociclib. d SMARCA2 restoration in SMARCA4/2-dual deficient cells H1703 confers resistance to palbociclib. Colony formation assay of H1703 cells expressing vector control or SMARCA2 and treated with palbociclib. e – g Resistance to palbociclib after restoration of SMARCA4 is also observed in mouse xenograft models using an isogenic cell pair of H1299 cells expressing vector control or SMARCA4 . e Tumor volume evolution during the course of the experiment in H1299 xenograft models expressing vector control (left) or SMARCA4 (right). f Tumor volume fold change during the establishment phase (left) and during palbociclib treatment (right) in the same models. g Immunohistochemistry (IHC) analysis of SMARCA4 in the representative endpoint tumors of H1703 control or SMARCA4-restored from above. Bar 50 µm. Error bars represent mean ± standard error of mean (s.e.m.); two-way analysis of variance (ANOVA), **** p

    Journal: Nature Communications

    Article Title: SMARCA4 loss is synthetic lethal with CDK4/6 inhibition in non-small cell lung cancer

    doi: 10.1038/s41467-019-08380-1

    Figure Lengend Snippet: SMARCA4 loss is synthetic lethal with cyclin-dependent kinase 4/6 (CDK4/6) inhibition in non-small cell lung cancer (NSCLC). a , b SMARCA4 restoration in SMARCA4-deficient cell lines confers drug resistance to palbociclib. Colony formation assays of H1299 ( a ) and H1703 ( b ) cells expressing vector control or SMARCA4 and treated with palbociclib (H1299, 300 nM; H1703, 100 nM). c SMARCA2 knockdown in SMARCA4-deficient H1299 cells sensitizes cells to palbociclib treatment. Colony formation assay of H1299 cells expressing pLKO control or SMARCA2 short hairpin RNAs (shRNAs) and treated with palbociclib. d SMARCA2 restoration in SMARCA4/2-dual deficient cells H1703 confers resistance to palbociclib. Colony formation assay of H1703 cells expressing vector control or SMARCA2 and treated with palbociclib. e – g Resistance to palbociclib after restoration of SMARCA4 is also observed in mouse xenograft models using an isogenic cell pair of H1299 cells expressing vector control or SMARCA4 . e Tumor volume evolution during the course of the experiment in H1299 xenograft models expressing vector control (left) or SMARCA4 (right). f Tumor volume fold change during the establishment phase (left) and during palbociclib treatment (right) in the same models. g Immunohistochemistry (IHC) analysis of SMARCA4 in the representative endpoint tumors of H1703 control or SMARCA4-restored from above. Bar 50 µm. Error bars represent mean ± standard error of mean (s.e.m.); two-way analysis of variance (ANOVA), **** p

    Article Snippet: IHC was performed on the TMAs using anti-SMARCA4 (anti-BRG1 antibody (clone EPNCIR111A, dilution, 1:100 dilution, Abcam, Cambridge, UK), anti-p16 (clone G175-405, 1:20 dilution, BD PharmingenTM ) and anti-RB1 (G3-245, 1:100 dilution, BD PharmingenTM ) antibodies.

    Techniques: Inhibition, Expressing, Plasmid Preparation, Colony Assay, Immunohistochemistry

    Reduced cyclin D1 in SMARCA4-deficient non-small cell lung cancer (NSCLC) cells causessensitivities to cyclin-dependent kinase 4/6 (CDK4/6) inhibitors. a , b SMARCA4-deficient NSCLC cell lines express reduced cyclin D1 levels. Western blot analysis for the indicated proteins ( a ) and CCND1 messenger RNA (mRNA) expression ( b ) of a panel of NSCLC cell lines. HSP90 was used as a loading control. Relative CCND1 mRNA expression (relative to GAPDH ) was measured by real-time quantitative reverse transcription PCR (RT-qPCR). A4: SMARCA4, A4/2: SMARCA4/2, Pro: proficient, Def: deficient, K: KRAS mutation. Empty triangles indicate RB-deficient cell lines. Turquoise color indicates cell lines with KRAS mutation. Error bars: mean ± standard deviation (s.d.) of biological replicates ( n = 3); two-tailed t test, * p

    Journal: Nature Communications

    Article Title: SMARCA4 loss is synthetic lethal with CDK4/6 inhibition in non-small cell lung cancer

    doi: 10.1038/s41467-019-08380-1

    Figure Lengend Snippet: Reduced cyclin D1 in SMARCA4-deficient non-small cell lung cancer (NSCLC) cells causessensitivities to cyclin-dependent kinase 4/6 (CDK4/6) inhibitors. a , b SMARCA4-deficient NSCLC cell lines express reduced cyclin D1 levels. Western blot analysis for the indicated proteins ( a ) and CCND1 messenger RNA (mRNA) expression ( b ) of a panel of NSCLC cell lines. HSP90 was used as a loading control. Relative CCND1 mRNA expression (relative to GAPDH ) was measured by real-time quantitative reverse transcription PCR (RT-qPCR). A4: SMARCA4, A4/2: SMARCA4/2, Pro: proficient, Def: deficient, K: KRAS mutation. Empty triangles indicate RB-deficient cell lines. Turquoise color indicates cell lines with KRAS mutation. Error bars: mean ± standard deviation (s.d.) of biological replicates ( n = 3); two-tailed t test, * p

    Article Snippet: IHC was performed on the TMAs using anti-SMARCA4 (anti-BRG1 antibody (clone EPNCIR111A, dilution, 1:100 dilution, Abcam, Cambridge, UK), anti-p16 (clone G175-405, 1:20 dilution, BD PharmingenTM ) and anti-RB1 (G3-245, 1:100 dilution, BD PharmingenTM ) antibodies.

    Techniques: Western Blot, Expressing, Polymerase Chain Reaction, Quantitative RT-PCR, Mutagenesis, Standard Deviation, Two Tailed Test

    Palbociclib is effective against SMARCA4-deficient non-small cell lung cancer (NSCLC) tumor growth in vivo. Palbociclib inhibits tumor growth in xenograft models of H1299 ( a , b , e , f ) and H1703 ( c , d , g , h ). a , c Tumor size from day 0 of treatment in H1299 ( a , n = 4 per group) and H1703 ( c , n = 8 for vehicle, n = 7 for palbociclib; 150 mg kg −1 ) models. Error bars represent mean ± standard error of mean (s.e.m.); two-way analysis of variance (ANOVA), **** p

    Journal: Nature Communications

    Article Title: SMARCA4 loss is synthetic lethal with CDK4/6 inhibition in non-small cell lung cancer

    doi: 10.1038/s41467-019-08380-1

    Figure Lengend Snippet: Palbociclib is effective against SMARCA4-deficient non-small cell lung cancer (NSCLC) tumor growth in vivo. Palbociclib inhibits tumor growth in xenograft models of H1299 ( a , b , e , f ) and H1703 ( c , d , g , h ). a , c Tumor size from day 0 of treatment in H1299 ( a , n = 4 per group) and H1703 ( c , n = 8 for vehicle, n = 7 for palbociclib; 150 mg kg −1 ) models. Error bars represent mean ± standard error of mean (s.e.m.); two-way analysis of variance (ANOVA), **** p

    Article Snippet: IHC was performed on the TMAs using anti-SMARCA4 (anti-BRG1 antibody (clone EPNCIR111A, dilution, 1:100 dilution, Abcam, Cambridge, UK), anti-p16 (clone G175-405, 1:20 dilution, BD PharmingenTM ) and anti-RB1 (G3-245, 1:100 dilution, BD PharmingenTM ) antibodies.

    Techniques: In Vivo

    Extensive opening of regulatory elements by induction of SMARCA4/2. a Venn diagram showing overlap of open chromatin sites in control infected H1703 cells with or without SMARCA4 overexpression. Note the dramatic increase in open chromatin sites upon SMARCA4 overexpression. b Distribution of SMARCA4-dependent and -independent open chromatin sites relative to nearest gene transcriptional start site. Note that SMARCA4-dependent sites are much less likely to be promoters (within 1 kb of transcription start site (TSS)). c , d Metaplot ( c ) and heatmap ( d ) of assay for transposase-accessible chromatin sequencing (ATAC-seq) read data from control-infected, SMARCA4-infected and SMARCA2-infected cells over the 62,878 SMARCA4-dependent ATAC peaks. Note similar effect of SMARCA2 and SMARCA4. e , f Metaplot ( e ) and heatmap ( f ) of H3K27Ac chromatin immunoprecipitation (ChIP) data from control-transfected, SMARCA4-infected and SMARCA2-infected cells over the 62,878 SMARCA4-dependent ATAC peaks

    Journal: Nature Communications

    Article Title: SMARCA4 loss is synthetic lethal with CDK4/6 inhibition in non-small cell lung cancer

    doi: 10.1038/s41467-019-08380-1

    Figure Lengend Snippet: Extensive opening of regulatory elements by induction of SMARCA4/2. a Venn diagram showing overlap of open chromatin sites in control infected H1703 cells with or without SMARCA4 overexpression. Note the dramatic increase in open chromatin sites upon SMARCA4 overexpression. b Distribution of SMARCA4-dependent and -independent open chromatin sites relative to nearest gene transcriptional start site. Note that SMARCA4-dependent sites are much less likely to be promoters (within 1 kb of transcription start site (TSS)). c , d Metaplot ( c ) and heatmap ( d ) of assay for transposase-accessible chromatin sequencing (ATAC-seq) read data from control-infected, SMARCA4-infected and SMARCA2-infected cells over the 62,878 SMARCA4-dependent ATAC peaks. Note similar effect of SMARCA2 and SMARCA4. e , f Metaplot ( e ) and heatmap ( f ) of H3K27Ac chromatin immunoprecipitation (ChIP) data from control-transfected, SMARCA4-infected and SMARCA2-infected cells over the 62,878 SMARCA4-dependent ATAC peaks

    Article Snippet: IHC was performed on the TMAs using anti-SMARCA4 (anti-BRG1 antibody (clone EPNCIR111A, dilution, 1:100 dilution, Abcam, Cambridge, UK), anti-p16 (clone G175-405, 1:20 dilution, BD PharmingenTM ) and anti-RB1 (G3-245, 1:100 dilution, BD PharmingenTM ) antibodies.

    Techniques: Infection, Over Expression, Sequencing, Chromatin Immunoprecipitation, Transfection

    SMARCA4/2 loss causes reduced cyclin D1 expression in non-small cell lung cancer (NSCLC). a – d SMARCA4/2 regulate cyclin D1 expression in NSCLC. a , b SMARCA4 restoration upregulates cyclin D1 protein (left) and messenger RNA (mRNA) (right) expression in H1299 ( a ) and H1703 ( b ) cells. c SMARCA2 knockdown in H1299 cells suppresses cyclin D1 protein (left) and mRNA (right) expression. d SMARCA2 restoration in H1703 cells elevates cyclin D1 protein (left) and mRNA (right) expression. Relative CCND1 mRNA expression (relative to GAPDH ) was measured by real-time quantitative reverse transcription PCR (RT-qPCR). Error bars: mean ± s.d. of biological replicates ( n = 3, two-tailed t -test, * p

    Journal: Nature Communications

    Article Title: SMARCA4 loss is synthetic lethal with CDK4/6 inhibition in non-small cell lung cancer

    doi: 10.1038/s41467-019-08380-1

    Figure Lengend Snippet: SMARCA4/2 loss causes reduced cyclin D1 expression in non-small cell lung cancer (NSCLC). a – d SMARCA4/2 regulate cyclin D1 expression in NSCLC. a , b SMARCA4 restoration upregulates cyclin D1 protein (left) and messenger RNA (mRNA) (right) expression in H1299 ( a ) and H1703 ( b ) cells. c SMARCA2 knockdown in H1299 cells suppresses cyclin D1 protein (left) and mRNA (right) expression. d SMARCA2 restoration in H1703 cells elevates cyclin D1 protein (left) and mRNA (right) expression. Relative CCND1 mRNA expression (relative to GAPDH ) was measured by real-time quantitative reverse transcription PCR (RT-qPCR). Error bars: mean ± s.d. of biological replicates ( n = 3, two-tailed t -test, * p

    Article Snippet: IHC was performed on the TMAs using anti-SMARCA4 (anti-BRG1 antibody (clone EPNCIR111A, dilution, 1:100 dilution, Abcam, Cambridge, UK), anti-p16 (clone G175-405, 1:20 dilution, BD PharmingenTM ) and anti-RB1 (G3-245, 1:100 dilution, BD PharmingenTM ) antibodies.

    Techniques: Expressing, Polymerase Chain Reaction, Quantitative RT-PCR, Two Tailed Test

    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

    BRG1 is required for hSNF5-mediated induction of p15 INK4b and p16 INK4a . (A) Western blot analysis of the BRG1 protein levels in MON cell extracts prepared 4 days after BRG1 knockdown using lentiviral transduction with viruses expressing shRNA targeting BRG1 mRNA (clone 15549; Open Biosystems; top panel, lanes 3 and 4). As a control, cells were transduced with lentiviruses expressing GFP. One day after transduction with either GFP- or shRNA-expressing lentiviruses, cells were transduced again with viruses expressing either GFP (middle panel, lanes 1 and 3) or hSNF5 (lanes 2 and 3). Cell extracts were prepared 72 h after hSNF5 expression. Histone H3 serves as a loading control. (B) Loss of BRG1 abrogates transcriptional activation of p15 INK4b and p16 INK4a by hSNF5. Relative expression levels of p15 INK4b , p14 ARF , and p16 INK4a in these cells were determined by RT-qPCR of isolated mRNA, 72 h after hSNF5 expression. The bar graphs represent the mean of three independent experiments, each analyzed in triplicate by RT-qPCR.

    Journal: Molecular and Cellular Biology

    Article Title: SWI/SNF Mediates Polycomb Eviction and Epigenetic Reprogramming of the INK4b-ARF-INK4a Locus

    doi: 10.1128/MCB.02019-07

    Figure Lengend Snippet: BRG1 is required for hSNF5-mediated induction of p15 INK4b and p16 INK4a . (A) Western blot analysis of the BRG1 protein levels in MON cell extracts prepared 4 days after BRG1 knockdown using lentiviral transduction with viruses expressing shRNA targeting BRG1 mRNA (clone 15549; Open Biosystems; top panel, lanes 3 and 4). As a control, cells were transduced with lentiviruses expressing GFP. One day after transduction with either GFP- or shRNA-expressing lentiviruses, cells were transduced again with viruses expressing either GFP (middle panel, lanes 1 and 3) or hSNF5 (lanes 2 and 3). Cell extracts were prepared 72 h after hSNF5 expression. Histone H3 serves as a loading control. (B) Loss of BRG1 abrogates transcriptional activation of p15 INK4b and p16 INK4a by hSNF5. Relative expression levels of p15 INK4b , p14 ARF , and p16 INK4a in these cells were determined by RT-qPCR of isolated mRNA, 72 h after hSNF5 expression. The bar graphs represent the mean of three independent experiments, each analyzed in triplicate by RT-qPCR.

    Article Snippet: The following antibodies were used: SNF5 (ab12167 [Abcam]), BRG1 (ab4081 [Abcam]), SUZ12 (ab12073 [Abcam]), BMI1 (ab14389 [Abcam]), EZH2 (Sc-25383 [Santa Cruz]), DNMT3B (IMG-184A [IMGENEX]), POL II (Sc-899 [Santa Cruz]), mixed lineage leukemia 1 (MLL1; A300-086A [Bethyl Laboratories]), Histone H3 (ab1791 [Abcam]), H3-K4me3 (ab12209 [Abcam]), and H3-K27me3 (catalog no. 07-449 [Upstate]).

    Techniques: Western Blot, Transduction, Expressing, shRNA, Activation Assay, Quantitative RT-PCR, Isolation

    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

    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

    Nucleosome eviction does not correlate with chromatin modification, BRG1 recruitment or H2A.Z deposition. The levels of H4Ac, H3Ac, H3K4Me3, BRG1 recruitment and H2A.Z deposition at the HLA-DRA promoter were analyzed by qChIP in wild-type Raji B cells (WT), CIITA-deficient B cells (CIITA −/− ) and RFX-deficient B cells (RFX −/− ). Results were corrected for nucleosome density using antibodies directed against unmodified histone H3, and were normalized relative to a control position at the TBP promoter. Results are expressed relative to the values observed in wild-type cells, and represent the means and standard deviations derived from three independent experiments. Primers used are indicated in Supplementary Table 1 .

    Journal: Nucleic Acids Research

    Article Title: Nucleosome eviction from MHC class II promoters controls positioning of the transcription start site

    doi: 10.1093/nar/gkp116

    Figure Lengend Snippet: Nucleosome eviction does not correlate with chromatin modification, BRG1 recruitment or H2A.Z deposition. The levels of H4Ac, H3Ac, H3K4Me3, BRG1 recruitment and H2A.Z deposition at the HLA-DRA promoter were analyzed by qChIP in wild-type Raji B cells (WT), CIITA-deficient B cells (CIITA −/− ) and RFX-deficient B cells (RFX −/− ). Results were corrected for nucleosome density using antibodies directed against unmodified histone H3, and were normalized relative to a control position at the TBP promoter. Results are expressed relative to the values observed in wild-type cells, and represent the means and standard deviations derived from three independent experiments. Primers used are indicated in Supplementary Table 1 .

    Article Snippet: BRG1 antibodies were obtained from Upstate (07-478) or Santacruz (sc-10768).

    Techniques: Modification, Derivative Assay

    BAF60a4-140 inhibits GR interaction with the BRG1 complex. (A) GR interaction with the BRG1 complex was examined by coimmunoprecipitation with an anti-BRG1 antibody. Input (100 μg of whole-cell extract) and the coimmunoprecipitated complex (from 1 mg of whole-cell extract) were analyzed by Western blotting with antibodies against BRG1, BAF155, GR, and BAF60a. (B) Models for BAF60a4-140 inhibition of GR interaction with the BRG1 complex. The BAF60a4-140 mutant may integrate into BRG1 complex alone or together with BAF60a to generate a nonfunctional complex. Alternatively, the BAF60a4-140 mutant may directly block the ability of the GR to interact with the complex.

    Journal: Molecular and Cellular Biology

    Article Title: BAF60a Mediates Critical Interactions between Nuclear Receptors and the BRG1 Chromatin-Remodeling Complex for Transactivation

    doi: 10.1128/MCB.23.17.6210-6220.2003

    Figure Lengend Snippet: BAF60a4-140 inhibits GR interaction with the BRG1 complex. (A) GR interaction with the BRG1 complex was examined by coimmunoprecipitation with an anti-BRG1 antibody. Input (100 μg of whole-cell extract) and the coimmunoprecipitated complex (from 1 mg of whole-cell extract) were analyzed by Western blotting with antibodies against BRG1, BAF155, GR, and BAF60a. (B) Models for BAF60a4-140 inhibition of GR interaction with the BRG1 complex. The BAF60a4-140 mutant may integrate into BRG1 complex alone or together with BAF60a to generate a nonfunctional complex. Alternatively, the BAF60a4-140 mutant may directly block the ability of the GR to interact with the complex.

    Article Snippet: Whole-cell extract (1 mg) was diluted into 0.5 ml of IP buffer containing 1% donkey serum (Sigma) and precleared once with 40 μl of protein G-agarose beads (Sigma) at 4°C for 1 h. Anti-BRG1 antibody (10 μg; N-15, Santa Cruz), was incubated with the lysate at 4°C overnight and then incubated with protein G-agarose for 1 h. After five washes with IP buffer, the immunoprecipitated complexes were eluted with 2× Laemmli SDS-PAGE sample buffer and boiled for 3 min. Coimmunoprecipitated proteins and input whole-cell extract (10%) were detected by Western blotting with antibodies against GR (against rat GR1-325), BRG1 (H88, Santa Cruz), BAF155 , and BAF60a (against BAF60a4-64).

    Techniques: Western Blot, Inhibition, Mutagenesis, Blocking Assay

    GR1-556 interacts with SRC-1 and BRG1 complex in vivo. (A) 2963.1 cells, which express endogenous PR, and derived 2963.1/556 cells were treated with ethanol (−), dexamethasone (Dex) (10 −7 M), or R5020 (10 −8 M) for 24 h. The CAT activity of cell lysate was analyzed by kinetic assays and normalized with total protein. The insert shows expression of GR1-556 in 2963.1/556 cells but not in the parental 2963.1 cells or A1-2 cells which express the GRwt. (B) SRC-1 associates with GR1-556 in 2963.1/556 cells. GR1-556-associated complexes were immunoprecipitated from whole-cell extracts of 2963.1/556 cells with no antibody (lane 3) or BUGR2 anti-GR antibody (lane 4) and immunoblotted with antibodies against SRC-1, BRG1, BAF155, and GR (BUGR2). The cognate inputs of SRC-1, BRG1, and BAF155 are shown in lanes 1 and 2 for the no-antibody (No Ab) and anti-GR antibody (αGR) immunoblots, respectively. (C) Interaction of GR1-556 with SRC-1, SRC-3, BRG1, BAF170, BAF155, BAF60a, and BAF57. GST and GR fusion proteins were used to pull down SRC-1, SRC-3, HDAC1, BRG1, and BAF170, 155, 60a, or 57. Input lysate (10%) was loaded as control.

    Journal: Molecular and Cellular Biology

    Article Title: BAF60a Mediates Critical Interactions between Nuclear Receptors and the BRG1 Chromatin-Remodeling Complex for Transactivation

    doi: 10.1128/MCB.23.17.6210-6220.2003

    Figure Lengend Snippet: GR1-556 interacts with SRC-1 and BRG1 complex in vivo. (A) 2963.1 cells, which express endogenous PR, and derived 2963.1/556 cells were treated with ethanol (−), dexamethasone (Dex) (10 −7 M), or R5020 (10 −8 M) for 24 h. The CAT activity of cell lysate was analyzed by kinetic assays and normalized with total protein. The insert shows expression of GR1-556 in 2963.1/556 cells but not in the parental 2963.1 cells or A1-2 cells which express the GRwt. (B) SRC-1 associates with GR1-556 in 2963.1/556 cells. GR1-556-associated complexes were immunoprecipitated from whole-cell extracts of 2963.1/556 cells with no antibody (lane 3) or BUGR2 anti-GR antibody (lane 4) and immunoblotted with antibodies against SRC-1, BRG1, BAF155, and GR (BUGR2). The cognate inputs of SRC-1, BRG1, and BAF155 are shown in lanes 1 and 2 for the no-antibody (No Ab) and anti-GR antibody (αGR) immunoblots, respectively. (C) Interaction of GR1-556 with SRC-1, SRC-3, BRG1, BAF170, BAF155, BAF60a, and BAF57. GST and GR fusion proteins were used to pull down SRC-1, SRC-3, HDAC1, BRG1, and BAF170, 155, 60a, or 57. Input lysate (10%) was loaded as control.

    Article Snippet: Whole-cell extract (1 mg) was diluted into 0.5 ml of IP buffer containing 1% donkey serum (Sigma) and precleared once with 40 μl of protein G-agarose beads (Sigma) at 4°C for 1 h. Anti-BRG1 antibody (10 μg; N-15, Santa Cruz), was incubated with the lysate at 4°C overnight and then incubated with protein G-agarose for 1 h. After five washes with IP buffer, the immunoprecipitated complexes were eluted with 2× Laemmli SDS-PAGE sample buffer and boiled for 3 min. Coimmunoprecipitated proteins and input whole-cell extract (10%) were detected by Western blotting with antibodies against GR (against rat GR1-325), BRG1 (H88, Santa Cruz), BAF155 , and BAF60a (against BAF60a4-64).

    Techniques: In Vivo, Derivative Assay, Activity Assay, Expressing, Immunoprecipitation, Western Blot

    GR(R488Q) mutant fails to interact with BAF60a in vitro and is unable to activate transcription from a chromatin template in vivo. (A) GRwt and GR(R488Q) were transfected with BRG1 and MMTV-CAT reporter into SW-13/M3-2 cells. Cells were treated posttransfection with ethanol (−) or dexamethasone (10 −7 M) (+) for 24 h. CAT and luciferase activities in the same cell lysate were plotted according to dexamethasone induction. (B) Expression of transfected BRG1, GRwt, and GR(R488Q) was examined by Western blotting.

    Journal: Molecular and Cellular Biology

    Article Title: BAF60a Mediates Critical Interactions between Nuclear Receptors and the BRG1 Chromatin-Remodeling Complex for Transactivation

    doi: 10.1128/MCB.23.17.6210-6220.2003

    Figure Lengend Snippet: GR(R488Q) mutant fails to interact with BAF60a in vitro and is unable to activate transcription from a chromatin template in vivo. (A) GRwt and GR(R488Q) were transfected with BRG1 and MMTV-CAT reporter into SW-13/M3-2 cells. Cells were treated posttransfection with ethanol (−) or dexamethasone (10 −7 M) (+) for 24 h. CAT and luciferase activities in the same cell lysate were plotted according to dexamethasone induction. (B) Expression of transfected BRG1, GRwt, and GR(R488Q) was examined by Western blotting.

    Article Snippet: Whole-cell extract (1 mg) was diluted into 0.5 ml of IP buffer containing 1% donkey serum (Sigma) and precleared once with 40 μl of protein G-agarose beads (Sigma) at 4°C for 1 h. Anti-BRG1 antibody (10 μg; N-15, Santa Cruz), was incubated with the lysate at 4°C overnight and then incubated with protein G-agarose for 1 h. After five washes with IP buffer, the immunoprecipitated complexes were eluted with 2× Laemmli SDS-PAGE sample buffer and boiled for 3 min. Coimmunoprecipitated proteins and input whole-cell extract (10%) were detected by Western blotting with antibodies against GR (against rat GR1-325), BRG1 (H88, Santa Cruz), BAF155 , and BAF60a (against BAF60a4-64).

    Techniques: Mutagenesis, In Vitro, In Vivo, Transfection, Luciferase, Expressing, Western Blot

    Both GR1-556 and the GR(R488Q) mutant exhibit decreased chromatin remodeling. (A) Chromatin remodeling stimulated by GRwt, GR(R488Q), or GR1-556 was examined by Sst I hypersensitivity. SW-13/M3-2 cells were transfected with BRG1 and GRwt, GR(R488Q), or GR1-556 for 24 h. Nuclei were isolated from the transfected cells after treatment with dexamethasone (10 −7 M) (+) or control (ethanol) (−) and were immediately subjected to limited digestion by Sst I to assess hypersensitivity of Nuc-B within the chromatin promoter. (B) The BRG1 complex interaction with GRwt, GR(R488Q), or GR1-556 in transfected SW-13/M3-2 cells was examined by coimmunoprecipitation with BRG1 antibody from 500 μg of whole-cell extract followed by Western blot analysis with antibodies against BRG1 and GR.

    Journal: Molecular and Cellular Biology

    Article Title: BAF60a Mediates Critical Interactions between Nuclear Receptors and the BRG1 Chromatin-Remodeling Complex for Transactivation

    doi: 10.1128/MCB.23.17.6210-6220.2003

    Figure Lengend Snippet: Both GR1-556 and the GR(R488Q) mutant exhibit decreased chromatin remodeling. (A) Chromatin remodeling stimulated by GRwt, GR(R488Q), or GR1-556 was examined by Sst I hypersensitivity. SW-13/M3-2 cells were transfected with BRG1 and GRwt, GR(R488Q), or GR1-556 for 24 h. Nuclei were isolated from the transfected cells after treatment with dexamethasone (10 −7 M) (+) or control (ethanol) (−) and were immediately subjected to limited digestion by Sst I to assess hypersensitivity of Nuc-B within the chromatin promoter. (B) The BRG1 complex interaction with GRwt, GR(R488Q), or GR1-556 in transfected SW-13/M3-2 cells was examined by coimmunoprecipitation with BRG1 antibody from 500 μg of whole-cell extract followed by Western blot analysis with antibodies against BRG1 and GR.

    Article Snippet: Whole-cell extract (1 mg) was diluted into 0.5 ml of IP buffer containing 1% donkey serum (Sigma) and precleared once with 40 μl of protein G-agarose beads (Sigma) at 4°C for 1 h. Anti-BRG1 antibody (10 μg; N-15, Santa Cruz), was incubated with the lysate at 4°C overnight and then incubated with protein G-agarose for 1 h. After five washes with IP buffer, the immunoprecipitated complexes were eluted with 2× Laemmli SDS-PAGE sample buffer and boiled for 3 min. Coimmunoprecipitated proteins and input whole-cell extract (10%) were detected by Western blotting with antibodies against GR (against rat GR1-325), BRG1 (H88, Santa Cruz), BAF155 , and BAF60a (against BAF60a4-64).

    Techniques: Mutagenesis, Transfection, Isolation, Western Blot

    Knockdown of BRG1 upregulates PTEN levels in DLD-1 cells. ( A ) Results of real-time quantitative RT–PCR analysis. The cyclophilin mRNA levels were examined as a quality and quantity of control of mRNA. * P

    Journal: British Journal of Cancer

    Article Title: Regulation of PTEN expression by the SWI/SNF chromatin-remodelling protein BRG1 in human colorectal carcinoma cells

    doi: 10.1038/sj.bjc.6606018

    Figure Lengend Snippet: Knockdown of BRG1 upregulates PTEN levels in DLD-1 cells. ( A ) Results of real-time quantitative RT–PCR analysis. The cyclophilin mRNA levels were examined as a quality and quantity of control of mRNA. * P

    Article Snippet: Antibodies against BRG1, BRM, E-cadherin (Santa Cruz), β -catenin (Cell Signaling), p-Akt, phospho-GSK-3β (Ser9) (p-GSK-3β , Cell Signaling) and cyclin D1 were used.

    Techniques: Quantitative RT-PCR

    The impact of BRG1 on the cyclin D1 levels via the PI3K–Akt signalling pathway in DLD-1 cells. ( A ) Results of western blot analysis. Cells were also treated with PI3K inhibitor LY294002. β -Actin was used as a loading control. ( B ) The status of p-Akt, p-GSK-3 β and cyclin D1 expressions in the BRG1 siRNA transfectant. ( C ) Transduction of recombinant Akt expression vector (p-Akt1) inhibited tumour-suppressive effects of BRG1 siRNA. β -Actin was used as a loading control. ( D ) Results of the growth test. * P

    Journal: British Journal of Cancer

    Article Title: Regulation of PTEN expression by the SWI/SNF chromatin-remodelling protein BRG1 in human colorectal carcinoma cells

    doi: 10.1038/sj.bjc.6606018

    Figure Lengend Snippet: The impact of BRG1 on the cyclin D1 levels via the PI3K–Akt signalling pathway in DLD-1 cells. ( A ) Results of western blot analysis. Cells were also treated with PI3K inhibitor LY294002. β -Actin was used as a loading control. ( B ) The status of p-Akt, p-GSK-3 β and cyclin D1 expressions in the BRG1 siRNA transfectant. ( C ) Transduction of recombinant Akt expression vector (p-Akt1) inhibited tumour-suppressive effects of BRG1 siRNA. β -Actin was used as a loading control. ( D ) Results of the growth test. * P

    Article Snippet: Antibodies against BRG1, BRM, E-cadherin (Santa Cruz), β -catenin (Cell Signaling), p-Akt, phospho-GSK-3β (Ser9) (p-GSK-3β , Cell Signaling) and cyclin D1 were used.

    Techniques: Western Blot, Transfection, Transduction, Recombinant, Expressing, Plasmid Preparation

    The status of BRG1 expression correlates with high cyclin D1 levels in CRC cases. ( A ) Representative illustrations of immunoreactivities against cyclin D1, PTEN and p-Akt antibodies. ( B ) Summary of immunohistochemical analyses. Immunoreactivities against BRG1, cyclin D1, PTEN and p-Akt were evaluated as described in the text.

    Journal: British Journal of Cancer

    Article Title: Regulation of PTEN expression by the SWI/SNF chromatin-remodelling protein BRG1 in human colorectal carcinoma cells

    doi: 10.1038/sj.bjc.6606018

    Figure Lengend Snippet: The status of BRG1 expression correlates with high cyclin D1 levels in CRC cases. ( A ) Representative illustrations of immunoreactivities against cyclin D1, PTEN and p-Akt antibodies. ( B ) Summary of immunohistochemical analyses. Immunoreactivities against BRG1, cyclin D1, PTEN and p-Akt were evaluated as described in the text.

    Article Snippet: Antibodies against BRG1, BRM, E-cadherin (Santa Cruz), β -catenin (Cell Signaling), p-Akt, phospho-GSK-3β (Ser9) (p-GSK-3β , Cell Signaling) and cyclin D1 were used.

    Techniques: Expressing, Immunohistochemistry

    The SWI/SNF chromatin-remodelling BRG1 and BRM expression in human CRC tissues. ( A ) Immunohistochemical results of BRG1 and BRM expression in the representative normal mucosa, adenoma and adenocarcinoma of the colorectum. ( B ) Average percentages of BRG1- and BRM-positive cells. * P

    Journal: British Journal of Cancer

    Article Title: Regulation of PTEN expression by the SWI/SNF chromatin-remodelling protein BRG1 in human colorectal carcinoma cells

    doi: 10.1038/sj.bjc.6606018

    Figure Lengend Snippet: The SWI/SNF chromatin-remodelling BRG1 and BRM expression in human CRC tissues. ( A ) Immunohistochemical results of BRG1 and BRM expression in the representative normal mucosa, adenoma and adenocarcinoma of the colorectum. ( B ) Average percentages of BRG1- and BRM-positive cells. * P

    Article Snippet: Antibodies against BRG1, BRM, E-cadherin (Santa Cruz), β -catenin (Cell Signaling), p-Akt, phospho-GSK-3β (Ser9) (p-GSK-3β , Cell Signaling) and cyclin D1 were used.

    Techniques: Expressing, Immunohistochemistry

    Effects of knockdown of BRG1 in human CRC cell lines. ( A ) Expressions of BRG1 and BRM in CRC cell lines. β -Actin was used as a loading control. ( B ) Nuclear localisation of BRG1 and BRM protein in DLD-1 cells. ( C ) Silencing of BRG1 by transduction of BRG1 siRNA into DLD-1 cells. The negative control siRNA was also transduced. ( D ) Results of cell growth test. * P

    Journal: British Journal of Cancer

    Article Title: Regulation of PTEN expression by the SWI/SNF chromatin-remodelling protein BRG1 in human colorectal carcinoma cells

    doi: 10.1038/sj.bjc.6606018

    Figure Lengend Snippet: Effects of knockdown of BRG1 in human CRC cell lines. ( A ) Expressions of BRG1 and BRM in CRC cell lines. β -Actin was used as a loading control. ( B ) Nuclear localisation of BRG1 and BRM protein in DLD-1 cells. ( C ) Silencing of BRG1 by transduction of BRG1 siRNA into DLD-1 cells. The negative control siRNA was also transduced. ( D ) Results of cell growth test. * P

    Article Snippet: Antibodies against BRG1, BRM, E-cadherin (Santa Cruz), β -catenin (Cell Signaling), p-Akt, phospho-GSK-3β (Ser9) (p-GSK-3β , Cell Signaling) and cyclin D1 were used.

    Techniques: Transduction, Negative Control

    SMARCA4/2 regulate CCND1 via controlling chromatin accessibility and upregulating JUN . a Assay for transposase-accessible chromatin sequencing (ATAC-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) data in vicinity of the CCND1 locus indicate enhanced chromatin accessibility upon SMARCA4/2 restoration. Note SMARCA4 at CCND1 promoter and formation of new putative enhancer ~50 kb upstream of CCND1 promoter. All data were generated in H1703 cells before and after restoration of SMARCA4 or SMARCA2, except the publicly available SMARCA4 ChIP data in H1299 cells expressing doxycycline (Dox)-inducible SMARCA4 34 . Track height is normalized to relative number of mapped reads. b Zoomed-in view of the putative CCND1 enhancer region. Shown are ATAC-seq peaks in H1703 cells before and after SMARCA4/2 restoration and the publicly available c-Fos/c-Jun ChIP data of endothelial cell line, human umbilical vein endothelial cell (HUVEC) (GSM935585, GSM935278). Location of canonical adaptor protein-1 (AP-1) motifs are indicated. c ATAC and ChIP-seq data in vicinity of JUN locus as described in a . Note SMARCA4 at JUN promoter and extensive opening of nearby putative enhancers. d– i Restoration of SMARCA4 in H1703 ( d , e ) and H1299 ( f , g ) or SMARCA2 restoration in H1703 ( h , i ) cells upregulate c-Jun messenger RNA (mRNA) ( d , f , h ) and protein ( e , g , i ). j , k Knockdown of JUN partially abrogated SMARCA4-mediated induction of cyclin D1 mRNA ( j ) and protein ( k ) expression in H1703 cells. l Proposed model showing that SMARCA4 directly regulates CCND1 and also upregulates JUN which positively regulates CCND1 . Two-tailed t -test. Error bars represent mean ± s.d., *** p

    Journal: Nature Communications

    Article Title: SMARCA4 loss is synthetic lethal with CDK4/6 inhibition in non-small cell lung cancer

    doi: 10.1038/s41467-019-08380-1

    Figure Lengend Snippet: SMARCA4/2 regulate CCND1 via controlling chromatin accessibility and upregulating JUN . a Assay for transposase-accessible chromatin sequencing (ATAC-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) data in vicinity of the CCND1 locus indicate enhanced chromatin accessibility upon SMARCA4/2 restoration. Note SMARCA4 at CCND1 promoter and formation of new putative enhancer ~50 kb upstream of CCND1 promoter. All data were generated in H1703 cells before and after restoration of SMARCA4 or SMARCA2, except the publicly available SMARCA4 ChIP data in H1299 cells expressing doxycycline (Dox)-inducible SMARCA4 34 . Track height is normalized to relative number of mapped reads. b Zoomed-in view of the putative CCND1 enhancer region. Shown are ATAC-seq peaks in H1703 cells before and after SMARCA4/2 restoration and the publicly available c-Fos/c-Jun ChIP data of endothelial cell line, human umbilical vein endothelial cell (HUVEC) (GSM935585, GSM935278). Location of canonical adaptor protein-1 (AP-1) motifs are indicated. c ATAC and ChIP-seq data in vicinity of JUN locus as described in a . Note SMARCA4 at JUN promoter and extensive opening of nearby putative enhancers. d– i Restoration of SMARCA4 in H1703 ( d , e ) and H1299 ( f , g ) or SMARCA2 restoration in H1703 ( h , i ) cells upregulate c-Jun messenger RNA (mRNA) ( d , f , h ) and protein ( e , g , i ). j , k Knockdown of JUN partially abrogated SMARCA4-mediated induction of cyclin D1 mRNA ( j ) and protein ( k ) expression in H1703 cells. l Proposed model showing that SMARCA4 directly regulates CCND1 and also upregulates JUN which positively regulates CCND1 . Two-tailed t -test. Error bars represent mean ± s.d., *** p

    Article Snippet: Antibodies against HSP90 (H-114), cyclin D1 (A12), CDK6 (C-21), CDK4 (DCS-35), p16 (C-20), p21 (H164), cyclin E (HE12), c-Jun (G4) and c-Fos (E8) were from Santa Cruz Biotechnology; antibodies against cyclin D2 (D52F9) and p-RB (S795) were from Cell Signaling; antibody against SMARCA4 were from Bethyl Laboratories (A300-813A).

    Techniques: Sequencing, ChIP-sequencing, Chromatin Immunoprecipitation, Generated, Expressing, Two Tailed Test

    SMARCA4 loss is synthetic lethal with cyclin-dependent kinase 4/6 (CDK4/6) inhibition in non-small cell lung cancer (NSCLC). a , b SMARCA4 restoration in SMARCA4-deficient cell lines confers drug resistance to palbociclib. Colony formation assays of H1299 ( a ) and H1703 ( b ) cells expressing vector control or SMARCA4 and treated with palbociclib (H1299, 300 nM; H1703, 100 nM). c SMARCA2 knockdown in SMARCA4-deficient H1299 cells sensitizes cells to palbociclib treatment. Colony formation assay of H1299 cells expressing pLKO control or SMARCA2 short hairpin RNAs (shRNAs) and treated with palbociclib. d SMARCA2 restoration in SMARCA4/2-dual deficient cells H1703 confers resistance to palbociclib. Colony formation assay of H1703 cells expressing vector control or SMARCA2 and treated with palbociclib. e – g Resistance to palbociclib after restoration of SMARCA4 is also observed in mouse xenograft models using an isogenic cell pair of H1299 cells expressing vector control or SMARCA4 . e Tumor volume evolution during the course of the experiment in H1299 xenograft models expressing vector control (left) or SMARCA4 (right). f Tumor volume fold change during the establishment phase (left) and during palbociclib treatment (right) in the same models. g Immunohistochemistry (IHC) analysis of SMARCA4 in the representative endpoint tumors of H1703 control or SMARCA4-restored from above. Bar 50 µm. Error bars represent mean ± standard error of mean (s.e.m.); two-way analysis of variance (ANOVA), **** p

    Journal: Nature Communications

    Article Title: SMARCA4 loss is synthetic lethal with CDK4/6 inhibition in non-small cell lung cancer

    doi: 10.1038/s41467-019-08380-1

    Figure Lengend Snippet: SMARCA4 loss is synthetic lethal with cyclin-dependent kinase 4/6 (CDK4/6) inhibition in non-small cell lung cancer (NSCLC). a , b SMARCA4 restoration in SMARCA4-deficient cell lines confers drug resistance to palbociclib. Colony formation assays of H1299 ( a ) and H1703 ( b ) cells expressing vector control or SMARCA4 and treated with palbociclib (H1299, 300 nM; H1703, 100 nM). c SMARCA2 knockdown in SMARCA4-deficient H1299 cells sensitizes cells to palbociclib treatment. Colony formation assay of H1299 cells expressing pLKO control or SMARCA2 short hairpin RNAs (shRNAs) and treated with palbociclib. d SMARCA2 restoration in SMARCA4/2-dual deficient cells H1703 confers resistance to palbociclib. Colony formation assay of H1703 cells expressing vector control or SMARCA2 and treated with palbociclib. e – g Resistance to palbociclib after restoration of SMARCA4 is also observed in mouse xenograft models using an isogenic cell pair of H1299 cells expressing vector control or SMARCA4 . e Tumor volume evolution during the course of the experiment in H1299 xenograft models expressing vector control (left) or SMARCA4 (right). f Tumor volume fold change during the establishment phase (left) and during palbociclib treatment (right) in the same models. g Immunohistochemistry (IHC) analysis of SMARCA4 in the representative endpoint tumors of H1703 control or SMARCA4-restored from above. Bar 50 µm. Error bars represent mean ± standard error of mean (s.e.m.); two-way analysis of variance (ANOVA), **** p

    Article Snippet: Antibodies against HSP90 (H-114), cyclin D1 (A12), CDK6 (C-21), CDK4 (DCS-35), p16 (C-20), p21 (H164), cyclin E (HE12), c-Jun (G4) and c-Fos (E8) were from Santa Cruz Biotechnology; antibodies against cyclin D2 (D52F9) and p-RB (S795) were from Cell Signaling; antibody against SMARCA4 were from Bethyl Laboratories (A300-813A).

    Techniques: Inhibition, Expressing, Plasmid Preparation, Colony Assay, Immunohistochemistry

    Reduced cyclin D1 in SMARCA4-deficient non-small cell lung cancer (NSCLC) cells causessensitivities to cyclin-dependent kinase 4/6 (CDK4/6) inhibitors. a , b SMARCA4-deficient NSCLC cell lines express reduced cyclin D1 levels. Western blot analysis for the indicated proteins ( a ) and CCND1 messenger RNA (mRNA) expression ( b ) of a panel of NSCLC cell lines. HSP90 was used as a loading control. Relative CCND1 mRNA expression (relative to GAPDH ) was measured by real-time quantitative reverse transcription PCR (RT-qPCR). A4: SMARCA4, A4/2: SMARCA4/2, Pro: proficient, Def: deficient, K: KRAS mutation. Empty triangles indicate RB-deficient cell lines. Turquoise color indicates cell lines with KRAS mutation. Error bars: mean ± standard deviation (s.d.) of biological replicates ( n = 3); two-tailed t test, * p

    Journal: Nature Communications

    Article Title: SMARCA4 loss is synthetic lethal with CDK4/6 inhibition in non-small cell lung cancer

    doi: 10.1038/s41467-019-08380-1

    Figure Lengend Snippet: Reduced cyclin D1 in SMARCA4-deficient non-small cell lung cancer (NSCLC) cells causessensitivities to cyclin-dependent kinase 4/6 (CDK4/6) inhibitors. a , b SMARCA4-deficient NSCLC cell lines express reduced cyclin D1 levels. Western blot analysis for the indicated proteins ( a ) and CCND1 messenger RNA (mRNA) expression ( b ) of a panel of NSCLC cell lines. HSP90 was used as a loading control. Relative CCND1 mRNA expression (relative to GAPDH ) was measured by real-time quantitative reverse transcription PCR (RT-qPCR). A4: SMARCA4, A4/2: SMARCA4/2, Pro: proficient, Def: deficient, K: KRAS mutation. Empty triangles indicate RB-deficient cell lines. Turquoise color indicates cell lines with KRAS mutation. Error bars: mean ± standard deviation (s.d.) of biological replicates ( n = 3); two-tailed t test, * p

    Article Snippet: Antibodies against HSP90 (H-114), cyclin D1 (A12), CDK6 (C-21), CDK4 (DCS-35), p16 (C-20), p21 (H164), cyclin E (HE12), c-Jun (G4) and c-Fos (E8) were from Santa Cruz Biotechnology; antibodies against cyclin D2 (D52F9) and p-RB (S795) were from Cell Signaling; antibody against SMARCA4 were from Bethyl Laboratories (A300-813A).

    Techniques: Western Blot, Expressing, Polymerase Chain Reaction, Quantitative RT-PCR, Mutagenesis, Standard Deviation, Two Tailed Test

    Palbociclib is effective against SMARCA4-deficient non-small cell lung cancer (NSCLC) tumor growth in vivo. Palbociclib inhibits tumor growth in xenograft models of H1299 ( a , b , e , f ) and H1703 ( c , d , g , h ). a , c Tumor size from day 0 of treatment in H1299 ( a , n = 4 per group) and H1703 ( c , n = 8 for vehicle, n = 7 for palbociclib; 150 mg kg −1 ) models. Error bars represent mean ± standard error of mean (s.e.m.); two-way analysis of variance (ANOVA), **** p

    Journal: Nature Communications

    Article Title: SMARCA4 loss is synthetic lethal with CDK4/6 inhibition in non-small cell lung cancer

    doi: 10.1038/s41467-019-08380-1

    Figure Lengend Snippet: Palbociclib is effective against SMARCA4-deficient non-small cell lung cancer (NSCLC) tumor growth in vivo. Palbociclib inhibits tumor growth in xenograft models of H1299 ( a , b , e , f ) and H1703 ( c , d , g , h ). a , c Tumor size from day 0 of treatment in H1299 ( a , n = 4 per group) and H1703 ( c , n = 8 for vehicle, n = 7 for palbociclib; 150 mg kg −1 ) models. Error bars represent mean ± standard error of mean (s.e.m.); two-way analysis of variance (ANOVA), **** p

    Article Snippet: Antibodies against HSP90 (H-114), cyclin D1 (A12), CDK6 (C-21), CDK4 (DCS-35), p16 (C-20), p21 (H164), cyclin E (HE12), c-Jun (G4) and c-Fos (E8) were from Santa Cruz Biotechnology; antibodies against cyclin D2 (D52F9) and p-RB (S795) were from Cell Signaling; antibody against SMARCA4 were from Bethyl Laboratories (A300-813A).

    Techniques: In Vivo

    Extensive opening of regulatory elements by induction of SMARCA4/2. a Venn diagram showing overlap of open chromatin sites in control infected H1703 cells with or without SMARCA4 overexpression. Note the dramatic increase in open chromatin sites upon SMARCA4 overexpression. b Distribution of SMARCA4-dependent and -independent open chromatin sites relative to nearest gene transcriptional start site. Note that SMARCA4-dependent sites are much less likely to be promoters (within 1 kb of transcription start site (TSS)). c , d Metaplot ( c ) and heatmap ( d ) of assay for transposase-accessible chromatin sequencing (ATAC-seq) read data from control-infected, SMARCA4-infected and SMARCA2-infected cells over the 62,878 SMARCA4-dependent ATAC peaks. Note similar effect of SMARCA2 and SMARCA4. e , f Metaplot ( e ) and heatmap ( f ) of H3K27Ac chromatin immunoprecipitation (ChIP) data from control-transfected, SMARCA4-infected and SMARCA2-infected cells over the 62,878 SMARCA4-dependent ATAC peaks

    Journal: Nature Communications

    Article Title: SMARCA4 loss is synthetic lethal with CDK4/6 inhibition in non-small cell lung cancer

    doi: 10.1038/s41467-019-08380-1

    Figure Lengend Snippet: Extensive opening of regulatory elements by induction of SMARCA4/2. a Venn diagram showing overlap of open chromatin sites in control infected H1703 cells with or without SMARCA4 overexpression. Note the dramatic increase in open chromatin sites upon SMARCA4 overexpression. b Distribution of SMARCA4-dependent and -independent open chromatin sites relative to nearest gene transcriptional start site. Note that SMARCA4-dependent sites are much less likely to be promoters (within 1 kb of transcription start site (TSS)). c , d Metaplot ( c ) and heatmap ( d ) of assay for transposase-accessible chromatin sequencing (ATAC-seq) read data from control-infected, SMARCA4-infected and SMARCA2-infected cells over the 62,878 SMARCA4-dependent ATAC peaks. Note similar effect of SMARCA2 and SMARCA4. e , f Metaplot ( e ) and heatmap ( f ) of H3K27Ac chromatin immunoprecipitation (ChIP) data from control-transfected, SMARCA4-infected and SMARCA2-infected cells over the 62,878 SMARCA4-dependent ATAC peaks

    Article Snippet: Antibodies against HSP90 (H-114), cyclin D1 (A12), CDK6 (C-21), CDK4 (DCS-35), p16 (C-20), p21 (H164), cyclin E (HE12), c-Jun (G4) and c-Fos (E8) were from Santa Cruz Biotechnology; antibodies against cyclin D2 (D52F9) and p-RB (S795) were from Cell Signaling; antibody against SMARCA4 were from Bethyl Laboratories (A300-813A).

    Techniques: Infection, Over Expression, Sequencing, Chromatin Immunoprecipitation, Transfection

    SMARCA4/2 loss causes reduced cyclin D1 expression in non-small cell lung cancer (NSCLC). a – d SMARCA4/2 regulate cyclin D1 expression in NSCLC. a , b SMARCA4 restoration upregulates cyclin D1 protein (left) and messenger RNA (mRNA) (right) expression in H1299 ( a ) and H1703 ( b ) cells. c SMARCA2 knockdown in H1299 cells suppresses cyclin D1 protein (left) and mRNA (right) expression. d SMARCA2 restoration in H1703 cells elevates cyclin D1 protein (left) and mRNA (right) expression. Relative CCND1 mRNA expression (relative to GAPDH ) was measured by real-time quantitative reverse transcription PCR (RT-qPCR). Error bars: mean ± s.d. of biological replicates ( n = 3, two-tailed t -test, * p

    Journal: Nature Communications

    Article Title: SMARCA4 loss is synthetic lethal with CDK4/6 inhibition in non-small cell lung cancer

    doi: 10.1038/s41467-019-08380-1

    Figure Lengend Snippet: SMARCA4/2 loss causes reduced cyclin D1 expression in non-small cell lung cancer (NSCLC). a – d SMARCA4/2 regulate cyclin D1 expression in NSCLC. a , b SMARCA4 restoration upregulates cyclin D1 protein (left) and messenger RNA (mRNA) (right) expression in H1299 ( a ) and H1703 ( b ) cells. c SMARCA2 knockdown in H1299 cells suppresses cyclin D1 protein (left) and mRNA (right) expression. d SMARCA2 restoration in H1703 cells elevates cyclin D1 protein (left) and mRNA (right) expression. Relative CCND1 mRNA expression (relative to GAPDH ) was measured by real-time quantitative reverse transcription PCR (RT-qPCR). Error bars: mean ± s.d. of biological replicates ( n = 3, two-tailed t -test, * p

    Article Snippet: Antibodies against HSP90 (H-114), cyclin D1 (A12), CDK6 (C-21), CDK4 (DCS-35), p16 (C-20), p21 (H164), cyclin E (HE12), c-Jun (G4) and c-Fos (E8) were from Santa Cruz Biotechnology; antibodies against cyclin D2 (D52F9) and p-RB (S795) were from Cell Signaling; antibody against SMARCA4 were from Bethyl Laboratories (A300-813A).

    Techniques: Expressing, Polymerase Chain Reaction, Quantitative RT-PCR, Two Tailed Test

    BRG1 does not alter p16 INK4a cell cycle regulation . A 50 μg of nuclear lysates from WMM1175_p16 INK4a clones with stably integrated siRNA targeting BRG1 or a non-specific (NS) control siRNA were probed for BRG1 and topoisomerase II (Topo II) as a loading control. B 50 μg of total cell lysates extracted from WMM1175_p16 INK4a cells stably expressing either a BRG1-specific siRNA or a non-specific (NS) siRNA molecule, as indicated, were treated with PBS (-) or IPTG (+) for 24 h and probed for p16 INK4a and β-actin. C Cell proliferation was determined by MTS assay. D A proportion of the IPTG/mock treated cells were analyzed for changes in cell cycle distribution. Percent S-phase change was calculated (percent S-phase mock treated cells – percent S-phase IPTG treated cells) × 100/percent S-phase mock treated cells. E The same clones were seeded at low density (10 3 cells/7.5 cm plate) and p16 INK4a expression was induced with 4 mM IPTG or cells mock treated and colony forming ability was assayed after 14 days.

    Journal: Molecular Cancer

    Article Title: The chromatin remodelling factor BRG1 is a novel binding partner of the tumor suppressor p16INK4a

    doi: 10.1186/1476-4598-8-4

    Figure Lengend Snippet: BRG1 does not alter p16 INK4a cell cycle regulation . A 50 μg of nuclear lysates from WMM1175_p16 INK4a clones with stably integrated siRNA targeting BRG1 or a non-specific (NS) control siRNA were probed for BRG1 and topoisomerase II (Topo II) as a loading control. B 50 μg of total cell lysates extracted from WMM1175_p16 INK4a cells stably expressing either a BRG1-specific siRNA or a non-specific (NS) siRNA molecule, as indicated, were treated with PBS (-) or IPTG (+) for 24 h and probed for p16 INK4a and β-actin. C Cell proliferation was determined by MTS assay. D A proportion of the IPTG/mock treated cells were analyzed for changes in cell cycle distribution. Percent S-phase change was calculated (percent S-phase mock treated cells – percent S-phase IPTG treated cells) × 100/percent S-phase mock treated cells. E The same clones were seeded at low density (10 3 cells/7.5 cm plate) and p16 INK4a expression was induced with 4 mM IPTG or cells mock treated and colony forming ability was assayed after 14 days.

    Article Snippet: Antibodies Mouse anti-β-actin (AC-74, Sigma, Castle Hill, NSW, Australia), mouse anti-Flag (M2, Sigma, Castle Hill, NSW, Australia), rabbit anti p16INK4a antibody (Western and immunohistochemistry, N-20, SantaCruz, Santa Cruz, CA, USA), mouse anti-p16INK4a antibody (immunoprecipitation, 2B4D11, Zymed Laboratories, San Francisco, CA, USA), mouse anti-BRG1 antibody (Western, G7, SantaCruz, Santa Cruz, CA, USA), rabbit anti-BRG1 antibody (immunohistochemistry, H-88, Santa Cruz, Santa Cruz, CA, USA), rabbit anti-MYC (A14, SantaCruz, Santa Cruz, CA, USA), Ki67 (MIB-1, Dako, Glostrup, Denmark), goat anti-BRM (Western, N-19, Santa Cruz, Santa Cruz, CA, USA), rabbit anti-BRM (immunohistochemistry, [ ]), mouse anti-CDK4 (C8218, Sigma, Castle Hill, NSW, Australia), mouse anti-CDK6 (MS-451-P0, Neomarker, Union City, CA, USA), rabbit anti-phosphorylated pRb (Ser807/811, Cell Signalling, Boston, MA, USA), mouse anti-pRb (G3-245, BD Pharmingen, Franklin Lakes, NJ, USA), mouse anti-topoisomerase II (Ab1, Oncogene, San Diego, CA, USA),

    Techniques: Clone Assay, Stable Transfection, Expressing, MTS Assay

    Immunohistochemistry of melanomas for BRM, p16 INK4a and BRG1 . Melanoma samples were stained for p16 INK4a and BRG1 with immunohistochemistry using DAB. BRM was stained using red fluorescence. Positive staining examples are presented in the right panel with no primary antibody control from the corresponding region in the left panel.

    Journal: Molecular Cancer

    Article Title: The chromatin remodelling factor BRG1 is a novel binding partner of the tumor suppressor p16INK4a

    doi: 10.1186/1476-4598-8-4

    Figure Lengend Snippet: Immunohistochemistry of melanomas for BRM, p16 INK4a and BRG1 . Melanoma samples were stained for p16 INK4a and BRG1 with immunohistochemistry using DAB. BRM was stained using red fluorescence. Positive staining examples are presented in the right panel with no primary antibody control from the corresponding region in the left panel.

    Article Snippet: Antibodies Mouse anti-β-actin (AC-74, Sigma, Castle Hill, NSW, Australia), mouse anti-Flag (M2, Sigma, Castle Hill, NSW, Australia), rabbit anti p16INK4a antibody (Western and immunohistochemistry, N-20, SantaCruz, Santa Cruz, CA, USA), mouse anti-p16INK4a antibody (immunoprecipitation, 2B4D11, Zymed Laboratories, San Francisco, CA, USA), mouse anti-BRG1 antibody (Western, G7, SantaCruz, Santa Cruz, CA, USA), rabbit anti-BRG1 antibody (immunohistochemistry, H-88, Santa Cruz, Santa Cruz, CA, USA), rabbit anti-MYC (A14, SantaCruz, Santa Cruz, CA, USA), Ki67 (MIB-1, Dako, Glostrup, Denmark), goat anti-BRM (Western, N-19, Santa Cruz, Santa Cruz, CA, USA), rabbit anti-BRM (immunohistochemistry, [ ]), mouse anti-CDK4 (C8218, Sigma, Castle Hill, NSW, Australia), mouse anti-CDK6 (MS-451-P0, Neomarker, Union City, CA, USA), rabbit anti-phosphorylated pRb (Ser807/811, Cell Signalling, Boston, MA, USA), mouse anti-pRb (G3-245, BD Pharmingen, Franklin Lakes, NJ, USA), mouse anti-topoisomerase II (Ab1, Oncogene, San Diego, CA, USA),

    Techniques: Immunohistochemistry, Staining, Fluorescence

    BRG1 binds p16 INK4a in melanoma cells and normal fibroblasts . A 50 μg of total cell lysates derived from uninduced (-) and induced (+) WMM1175_p16 INK4a cells and WS-1 fibroblasts (passage 20) were separated using a 15% SDS-PAGE gel. Immunoblots were probed for p16 INK4a and β-actin as indicated. B WMM1175_p16 INK4a cells were induced to express p16 INK4a with 4 mM IPTG or mock treated for 72 hours. Immunoprecipitations were performed using a mouse anti-p16 INK4a antibody or a matched mouse IgG from nuclear cell lysate, as indicated. Immunoblots were probed for endogenous BRG1 and induced p16 INK4a using a mouse anti-BRG1 and rabbit anti-p16 INK4a , respectively. C Endogenous BRG1 was co-immunoprecipitated with p16 INK4a from WS-1 normal dermal human fibroblasts grown to passage 20 as detailed above.

    Journal: Molecular Cancer

    Article Title: The chromatin remodelling factor BRG1 is a novel binding partner of the tumor suppressor p16INK4a

    doi: 10.1186/1476-4598-8-4

    Figure Lengend Snippet: BRG1 binds p16 INK4a in melanoma cells and normal fibroblasts . A 50 μg of total cell lysates derived from uninduced (-) and induced (+) WMM1175_p16 INK4a cells and WS-1 fibroblasts (passage 20) were separated using a 15% SDS-PAGE gel. Immunoblots were probed for p16 INK4a and β-actin as indicated. B WMM1175_p16 INK4a cells were induced to express p16 INK4a with 4 mM IPTG or mock treated for 72 hours. Immunoprecipitations were performed using a mouse anti-p16 INK4a antibody or a matched mouse IgG from nuclear cell lysate, as indicated. Immunoblots were probed for endogenous BRG1 and induced p16 INK4a using a mouse anti-BRG1 and rabbit anti-p16 INK4a , respectively. C Endogenous BRG1 was co-immunoprecipitated with p16 INK4a from WS-1 normal dermal human fibroblasts grown to passage 20 as detailed above.

    Article Snippet: Antibodies Mouse anti-β-actin (AC-74, Sigma, Castle Hill, NSW, Australia), mouse anti-Flag (M2, Sigma, Castle Hill, NSW, Australia), rabbit anti p16INK4a antibody (Western and immunohistochemistry, N-20, SantaCruz, Santa Cruz, CA, USA), mouse anti-p16INK4a antibody (immunoprecipitation, 2B4D11, Zymed Laboratories, San Francisco, CA, USA), mouse anti-BRG1 antibody (Western, G7, SantaCruz, Santa Cruz, CA, USA), rabbit anti-BRG1 antibody (immunohistochemistry, H-88, Santa Cruz, Santa Cruz, CA, USA), rabbit anti-MYC (A14, SantaCruz, Santa Cruz, CA, USA), Ki67 (MIB-1, Dako, Glostrup, Denmark), goat anti-BRM (Western, N-19, Santa Cruz, Santa Cruz, CA, USA), rabbit anti-BRM (immunohistochemistry, [ ]), mouse anti-CDK4 (C8218, Sigma, Castle Hill, NSW, Australia), mouse anti-CDK6 (MS-451-P0, Neomarker, Union City, CA, USA), rabbit anti-phosphorylated pRb (Ser807/811, Cell Signalling, Boston, MA, USA), mouse anti-pRb (G3-245, BD Pharmingen, Franklin Lakes, NJ, USA), mouse anti-topoisomerase II (Ab1, Oncogene, San Diego, CA, USA),

    Techniques: Derivative Assay, SDS Page, Western Blot, Immunoprecipitation

    pRb pathway proteins in cell lines . Expression of BRG1 and BRM was analyzed using 50 μg of nuclear cell lysates. All other proteins were analyzed from 50 μg of total cell lysates.

    Journal: Molecular Cancer

    Article Title: The chromatin remodelling factor BRG1 is a novel binding partner of the tumor suppressor p16INK4a

    doi: 10.1186/1476-4598-8-4

    Figure Lengend Snippet: pRb pathway proteins in cell lines . Expression of BRG1 and BRM was analyzed using 50 μg of nuclear cell lysates. All other proteins were analyzed from 50 μg of total cell lysates.

    Article Snippet: Antibodies Mouse anti-β-actin (AC-74, Sigma, Castle Hill, NSW, Australia), mouse anti-Flag (M2, Sigma, Castle Hill, NSW, Australia), rabbit anti p16INK4a antibody (Western and immunohistochemistry, N-20, SantaCruz, Santa Cruz, CA, USA), mouse anti-p16INK4a antibody (immunoprecipitation, 2B4D11, Zymed Laboratories, San Francisco, CA, USA), mouse anti-BRG1 antibody (Western, G7, SantaCruz, Santa Cruz, CA, USA), rabbit anti-BRG1 antibody (immunohistochemistry, H-88, Santa Cruz, Santa Cruz, CA, USA), rabbit anti-MYC (A14, SantaCruz, Santa Cruz, CA, USA), Ki67 (MIB-1, Dako, Glostrup, Denmark), goat anti-BRM (Western, N-19, Santa Cruz, Santa Cruz, CA, USA), rabbit anti-BRM (immunohistochemistry, [ ]), mouse anti-CDK4 (C8218, Sigma, Castle Hill, NSW, Australia), mouse anti-CDK6 (MS-451-P0, Neomarker, Union City, CA, USA), rabbit anti-phosphorylated pRb (Ser807/811, Cell Signalling, Boston, MA, USA), mouse anti-pRb (G3-245, BD Pharmingen, Franklin Lakes, NJ, USA), mouse anti-topoisomerase II (Ab1, Oncogene, San Diego, CA, USA),

    Techniques: Expressing

    BRG1 and p16 INK4a in cell cycle regulation . Indicated cell lines were transfected with MYC-p16 INK4a , FLAG-BRG1 and/or a control vector plus GFP-spectrin. Cells were fixed with 70% ethanol 48 hours post transfection and cellular DNA was stained with propidium iodide. Percent S-phase change of GFP-spectrin positive cells was calculated (percent S-phase vector control - percent S-phase sample) × 100/percent S-phase vector control.

    Journal: Molecular Cancer

    Article Title: The chromatin remodelling factor BRG1 is a novel binding partner of the tumor suppressor p16INK4a

    doi: 10.1186/1476-4598-8-4

    Figure Lengend Snippet: BRG1 and p16 INK4a in cell cycle regulation . Indicated cell lines were transfected with MYC-p16 INK4a , FLAG-BRG1 and/or a control vector plus GFP-spectrin. Cells were fixed with 70% ethanol 48 hours post transfection and cellular DNA was stained with propidium iodide. Percent S-phase change of GFP-spectrin positive cells was calculated (percent S-phase vector control - percent S-phase sample) × 100/percent S-phase vector control.

    Article Snippet: Antibodies Mouse anti-β-actin (AC-74, Sigma, Castle Hill, NSW, Australia), mouse anti-Flag (M2, Sigma, Castle Hill, NSW, Australia), rabbit anti p16INK4a antibody (Western and immunohistochemistry, N-20, SantaCruz, Santa Cruz, CA, USA), mouse anti-p16INK4a antibody (immunoprecipitation, 2B4D11, Zymed Laboratories, San Francisco, CA, USA), mouse anti-BRG1 antibody (Western, G7, SantaCruz, Santa Cruz, CA, USA), rabbit anti-BRG1 antibody (immunohistochemistry, H-88, Santa Cruz, Santa Cruz, CA, USA), rabbit anti-MYC (A14, SantaCruz, Santa Cruz, CA, USA), Ki67 (MIB-1, Dako, Glostrup, Denmark), goat anti-BRM (Western, N-19, Santa Cruz, Santa Cruz, CA, USA), rabbit anti-BRM (immunohistochemistry, [ ]), mouse anti-CDK4 (C8218, Sigma, Castle Hill, NSW, Australia), mouse anti-CDK6 (MS-451-P0, Neomarker, Union City, CA, USA), rabbit anti-phosphorylated pRb (Ser807/811, Cell Signalling, Boston, MA, USA), mouse anti-pRb (G3-245, BD Pharmingen, Franklin Lakes, NJ, USA), mouse anti-topoisomerase II (Ab1, Oncogene, San Diego, CA, USA),

    Techniques: Transfection, Plasmid Preparation, Staining

    BRG1 does not alter p16 INK4a driven senescence . WMM1175_p16 INK4a cells, BRG1 silenced (clone X1, left panel) or NS (clone E1, right panel), were exposed to 4 mM IPTG over a five-day period and analyzed by FACS analysis, Western blot and imunocytostaining: A 50 μg of total cell lysate were immunoblotted and probed for p16 INK4a , phospho-pRb (pRbSer 807/811 ) and as a loading control β-actin. B The accumulation of p16 INK4a , the cell proliferation marker Ki67, chromatin condensation (DAPI) and the appearance of SA-β-gal was analyzed by immunocytostaining in WMM1175_p16 INK4a . Enlarged images of cells (indicated with arrows) show DAPI-stained chromatin foci. Histograms correspond to the average ± s.d of at least two independent induction experiments from a total of at least 500 cells. LM, light microscopy. C FACS analysis by Forward Scatter (FSC) and Side Scatter (SSC) of clones demonstrate the senescence associated increase of cell size (FSC) and granularity (SSC) upon p16 INK4a induction.

    Journal: Molecular Cancer

    Article Title: The chromatin remodelling factor BRG1 is a novel binding partner of the tumor suppressor p16INK4a

    doi: 10.1186/1476-4598-8-4

    Figure Lengend Snippet: BRG1 does not alter p16 INK4a driven senescence . WMM1175_p16 INK4a cells, BRG1 silenced (clone X1, left panel) or NS (clone E1, right panel), were exposed to 4 mM IPTG over a five-day period and analyzed by FACS analysis, Western blot and imunocytostaining: A 50 μg of total cell lysate were immunoblotted and probed for p16 INK4a , phospho-pRb (pRbSer 807/811 ) and as a loading control β-actin. B The accumulation of p16 INK4a , the cell proliferation marker Ki67, chromatin condensation (DAPI) and the appearance of SA-β-gal was analyzed by immunocytostaining in WMM1175_p16 INK4a . Enlarged images of cells (indicated with arrows) show DAPI-stained chromatin foci. Histograms correspond to the average ± s.d of at least two independent induction experiments from a total of at least 500 cells. LM, light microscopy. C FACS analysis by Forward Scatter (FSC) and Side Scatter (SSC) of clones demonstrate the senescence associated increase of cell size (FSC) and granularity (SSC) upon p16 INK4a induction.

    Article Snippet: Antibodies Mouse anti-β-actin (AC-74, Sigma, Castle Hill, NSW, Australia), mouse anti-Flag (M2, Sigma, Castle Hill, NSW, Australia), rabbit anti p16INK4a antibody (Western and immunohistochemistry, N-20, SantaCruz, Santa Cruz, CA, USA), mouse anti-p16INK4a antibody (immunoprecipitation, 2B4D11, Zymed Laboratories, San Francisco, CA, USA), mouse anti-BRG1 antibody (Western, G7, SantaCruz, Santa Cruz, CA, USA), rabbit anti-BRG1 antibody (immunohistochemistry, H-88, Santa Cruz, Santa Cruz, CA, USA), rabbit anti-MYC (A14, SantaCruz, Santa Cruz, CA, USA), Ki67 (MIB-1, Dako, Glostrup, Denmark), goat anti-BRM (Western, N-19, Santa Cruz, Santa Cruz, CA, USA), rabbit anti-BRM (immunohistochemistry, [ ]), mouse anti-CDK4 (C8218, Sigma, Castle Hill, NSW, Australia), mouse anti-CDK6 (MS-451-P0, Neomarker, Union City, CA, USA), rabbit anti-phosphorylated pRb (Ser807/811, Cell Signalling, Boston, MA, USA), mouse anti-pRb (G3-245, BD Pharmingen, Franklin Lakes, NJ, USA), mouse anti-topoisomerase II (Ab1, Oncogene, San Diego, CA, USA),

    Techniques: FACS, Western Blot, Marker, Staining, Light Microscopy, Clone Assay

    Identification of BRG1 as p16 INK4a binding partner . A Schematic illustration of BRG1 highlighting the domains isolated in the yeast 2-hybrid screen (Y2H clone) B U2OS cells were transfected with MYC-p16 INK4a and FLAG-BRG1 or control vector and immunoprecipitations were performed with a mouse-anti-FLAG antibody or a matched mouse IgG as indicated. BRG1 and p16 INK4a were detected on immunoblots with anti-FLAG and anti-MYC antibodies. C Fluorescent microscopy images (FM) and confocal microscopy images (CF) of SW-13 cells grown on cover slips and transfected with MYC-p16 INK4a and FLAG-BRG1 and probed with anti-FLAG and anti-MYC antibodies.

    Journal: Molecular Cancer

    Article Title: The chromatin remodelling factor BRG1 is a novel binding partner of the tumor suppressor p16INK4a

    doi: 10.1186/1476-4598-8-4

    Figure Lengend Snippet: Identification of BRG1 as p16 INK4a binding partner . A Schematic illustration of BRG1 highlighting the domains isolated in the yeast 2-hybrid screen (Y2H clone) B U2OS cells were transfected with MYC-p16 INK4a and FLAG-BRG1 or control vector and immunoprecipitations were performed with a mouse-anti-FLAG antibody or a matched mouse IgG as indicated. BRG1 and p16 INK4a were detected on immunoblots with anti-FLAG and anti-MYC antibodies. C Fluorescent microscopy images (FM) and confocal microscopy images (CF) of SW-13 cells grown on cover slips and transfected with MYC-p16 INK4a and FLAG-BRG1 and probed with anti-FLAG and anti-MYC antibodies.

    Article Snippet: Antibodies Mouse anti-β-actin (AC-74, Sigma, Castle Hill, NSW, Australia), mouse anti-Flag (M2, Sigma, Castle Hill, NSW, Australia), rabbit anti p16INK4a antibody (Western and immunohistochemistry, N-20, SantaCruz, Santa Cruz, CA, USA), mouse anti-p16INK4a antibody (immunoprecipitation, 2B4D11, Zymed Laboratories, San Francisco, CA, USA), mouse anti-BRG1 antibody (Western, G7, SantaCruz, Santa Cruz, CA, USA), rabbit anti-BRG1 antibody (immunohistochemistry, H-88, Santa Cruz, Santa Cruz, CA, USA), rabbit anti-MYC (A14, SantaCruz, Santa Cruz, CA, USA), Ki67 (MIB-1, Dako, Glostrup, Denmark), goat anti-BRM (Western, N-19, Santa Cruz, Santa Cruz, CA, USA), rabbit anti-BRM (immunohistochemistry, [ ]), mouse anti-CDK4 (C8218, Sigma, Castle Hill, NSW, Australia), mouse anti-CDK6 (MS-451-P0, Neomarker, Union City, CA, USA), rabbit anti-phosphorylated pRb (Ser807/811, Cell Signalling, Boston, MA, USA), mouse anti-pRb (G3-245, BD Pharmingen, Franklin Lakes, NJ, USA), mouse anti-topoisomerase II (Ab1, Oncogene, San Diego, CA, USA),

    Techniques: Binding Assay, Isolation, Transfection, Plasmid Preparation, Western Blot, Microscopy, Confocal Microscopy

    Telomerase reverse transcriptase (TERT) serves as a transcriptional modulator to regulate FASL expression in Bone marrow mesenchymal stem cells (BMMSCs). A–B Western blot analysis showed decreased levels of FASL and active β-catenin, but not BRG1, in TERT − / − BMMSCs (A) and tert knockdown BMMSCs by siRNA (B) compared to TERT +/+ (WT) BMMSCs. C β-catenin activator (Chir, 10 μM) treatment elevated levels of active β-catenin and FASL in WT BMMSCs. fasl knockdown BMMSCs by siRNA showed a decreased level of FASL expression, but not active β-catenin. D In vitro coculture system showed β-catenin activator (Chir)-treated BMMSCs had increased capacity to induce AnnexinV + 7AAD − and AnnexinV + 7AAD + double positive apoptotic T cells compared to control group. fasl siRNA treatment could reduce Chir-elevated T cell apoptosis in the co-culture system. E Telomerase activity in Chir-treated BMMSCs showed no significant difference from the untreated group. 293T cells were used as a positive control, and heat-inactivated (H.I.) samples were used as a negative control. F Western blot analysis showed decreased expression levels of β-catenin and FASL in β-catenin knockdown BMMSCs by siRNA. G β-catenin knockdown BMMSCs by siRNA showed decreased capacity to induce AnnexinV + 7AAD − and AnnexinV + 7AAD + double positive apoptotic T cells compared to the control siRNA group. H Western blot showed that TERT − / − BMMSCs decreased expression levels of TERT, active β-catenin, and FASL. Tert transfection (TERT TF) rescued the expression levels of TERT, active β-catenin, and FASL, assessed by Western blot, while fasl transfection (FASL TF) only rescued FASL expression, but not that of TERT or β-catenin, in TERT − / − BMMSCs. I In vitro coculture system showed a decreased capacity of TERT − / − BMMSCs to induce AnnexinV + 7AAD − and AnnexinV + 7AAD + double positive apoptotic T cells when compared to the control group, whereas transfection of both tert and fasl rescued the capacity to induce AnnexinV + 7AAD − and AnnexinV + 7AAD + double positive apoptotic T cells. J fasl promoter-luciferase fusions were examined in WT, TERT − / − and TERT TF BMMSCs. Promoter activity was expressed as relative light units (RLU) normalized to the activity of co-transfected Renilla luciferase. The activity of 2 kb promoter-luciferase fusion was significantly elevated compared to 1.1 kb fusion in WT BMMSCs and TERT TF BMMSCs when compared to TERT − / − BMMSCs. The activity of TBE-specific site-mutated promoters was markedly decreased in WT BMMSCs and TERT TF BMMSCs. K Chromatin immunoprecipitation (ChIP)-qPCR assay showed enrichment of direct association of β-catenin on the fasl promoter in WT and TERT − / − BMMSCs, while the enrichment of direct association of TERT on the fasl promoter was only found in WT BMMSCs. L ChIP-Western blot assays showed direct association of TERT, β-catenin and BRG1 on the fasl promoter in WT BMMSCs, but only direct association of β-catenin and BRG1 on the fasl promoter in TERT − / − BMMSCs. M Schematic diagram indicates that TERT, as a transcriptional modulator in a complex with β-catenin and BRG1, mediates FASL expression in BMMSC-induced immunoregulation. Vehicle: scrambled siRNA-treated BMMSCs. Data information: Error bars represent the s.d. from the mean values (One-way ANOVA, Bonferroni, n = 3 in each group, *** P

    Journal: EMBO Molecular Medicine

    Article Title: Telomerase governs immunomodulatory properties of mesenchymal stem cells by regulating FAS ligand expression

    doi: 10.1002/emmm.201303000

    Figure Lengend Snippet: Telomerase reverse transcriptase (TERT) serves as a transcriptional modulator to regulate FASL expression in Bone marrow mesenchymal stem cells (BMMSCs). A–B Western blot analysis showed decreased levels of FASL and active β-catenin, but not BRG1, in TERT − / − BMMSCs (A) and tert knockdown BMMSCs by siRNA (B) compared to TERT +/+ (WT) BMMSCs. C β-catenin activator (Chir, 10 μM) treatment elevated levels of active β-catenin and FASL in WT BMMSCs. fasl knockdown BMMSCs by siRNA showed a decreased level of FASL expression, but not active β-catenin. D In vitro coculture system showed β-catenin activator (Chir)-treated BMMSCs had increased capacity to induce AnnexinV + 7AAD − and AnnexinV + 7AAD + double positive apoptotic T cells compared to control group. fasl siRNA treatment could reduce Chir-elevated T cell apoptosis in the co-culture system. E Telomerase activity in Chir-treated BMMSCs showed no significant difference from the untreated group. 293T cells were used as a positive control, and heat-inactivated (H.I.) samples were used as a negative control. F Western blot analysis showed decreased expression levels of β-catenin and FASL in β-catenin knockdown BMMSCs by siRNA. G β-catenin knockdown BMMSCs by siRNA showed decreased capacity to induce AnnexinV + 7AAD − and AnnexinV + 7AAD + double positive apoptotic T cells compared to the control siRNA group. H Western blot showed that TERT − / − BMMSCs decreased expression levels of TERT, active β-catenin, and FASL. Tert transfection (TERT TF) rescued the expression levels of TERT, active β-catenin, and FASL, assessed by Western blot, while fasl transfection (FASL TF) only rescued FASL expression, but not that of TERT or β-catenin, in TERT − / − BMMSCs. I In vitro coculture system showed a decreased capacity of TERT − / − BMMSCs to induce AnnexinV + 7AAD − and AnnexinV + 7AAD + double positive apoptotic T cells when compared to the control group, whereas transfection of both tert and fasl rescued the capacity to induce AnnexinV + 7AAD − and AnnexinV + 7AAD + double positive apoptotic T cells. J fasl promoter-luciferase fusions were examined in WT, TERT − / − and TERT TF BMMSCs. Promoter activity was expressed as relative light units (RLU) normalized to the activity of co-transfected Renilla luciferase. The activity of 2 kb promoter-luciferase fusion was significantly elevated compared to 1.1 kb fusion in WT BMMSCs and TERT TF BMMSCs when compared to TERT − / − BMMSCs. The activity of TBE-specific site-mutated promoters was markedly decreased in WT BMMSCs and TERT TF BMMSCs. K Chromatin immunoprecipitation (ChIP)-qPCR assay showed enrichment of direct association of β-catenin on the fasl promoter in WT and TERT − / − BMMSCs, while the enrichment of direct association of TERT on the fasl promoter was only found in WT BMMSCs. L ChIP-Western blot assays showed direct association of TERT, β-catenin and BRG1 on the fasl promoter in WT BMMSCs, but only direct association of β-catenin and BRG1 on the fasl promoter in TERT − / − BMMSCs. M Schematic diagram indicates that TERT, as a transcriptional modulator in a complex with β-catenin and BRG1, mediates FASL expression in BMMSC-induced immunoregulation. Vehicle: scrambled siRNA-treated BMMSCs. Data information: Error bars represent the s.d. from the mean values (One-way ANOVA, Bonferroni, n = 3 in each group, *** P

    Article Snippet: Anti-BRG1 antibody was purchased from Cell Signaling (Danvers, MA, USA).

    Techniques: Expressing, Western Blot, In Vitro, Co-Culture Assay, Activity Assay, Positive Control, Negative Control, Transfection, Luciferase, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Arsenite-induced HSF1 transcription requires BRG1, but not HLTF or SSRP1, in MCF7 cells. A , representative immunoblots showing knockdown levels of the indicated factors with their corresponding siRNA and shRNA. B , qRT-PCR analysis showing the levels of HSF1 mRNA by arsenite treatment with the indicated factors down-regulated. Error bar , mean ± S.D. ( n = 3); ***, p

    Journal: The Journal of Biological Chemistry

    Article Title: NRF2 transcriptionally activates the heat shock factor 1 promoter under oxidative stress and affects survival and migration potential of MCF7 cells

    doi: 10.1074/jbc.RA118.003376

    Figure Lengend Snippet: Arsenite-induced HSF1 transcription requires BRG1, but not HLTF or SSRP1, in MCF7 cells. A , representative immunoblots showing knockdown levels of the indicated factors with their corresponding siRNA and shRNA. B , qRT-PCR analysis showing the levels of HSF1 mRNA by arsenite treatment with the indicated factors down-regulated. Error bar , mean ± S.D. ( n = 3); ***, p

    Article Snippet: The antibodies used were anti-HSF1 (BioBharati LifeSciences, AB 0220), anti-HSP70 (BioBharati LifeSciences, AB 0210), anti-NRF2 (Cell Signaling Technology, 12721), anti–β-actin HRP secondary (Abcam, ab20272), anti–HO-1 (Cell Signaling Technology, 5853), anti-HLTF (SMARCA3) (Novus, NB100–280), anti-BRG1 (Cell Signaling Technology, 3508), anti-SSRP1 (BioLegend, 609702), anti–E-cadherin (Cell Signaling Technology, 3195), anti–N-cadherin (Cell Signaling Technology, 13116), anti-ATG7 (Cell Signaling Technology, 2631), and anti-LC3B (Cell Signaling Technology, 3868).

    Techniques: Western Blot, shRNA, Quantitative RT-PCR

    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

    β-actin is required for the regulation of chromatin status and the expression of Zic and Irx genes in MEFs or CiNeurons. (A) The relative expression of Zic amd Irx genes in MEFs and CiNeurons. Data are summary of at least 3 biological replicates from RNA-seq analysis. Error bar: S.E.M. (B) H3K9Me3 and Brg1 ChIP-seq analysis in WTM and KOM cells at Zic and Irx loci. Normalized signal at Zic1 ; Zic2 ; Zic3 ; Zic4 ; Irx1 ; and Irx3 loci were shown. The y-axis data range represents RPKM (Reads Per Kilobase of sequence range per Million mapped reads) per bin. The y-axis of tracks in the same image were set as the same range. Gene body position (exon: box, intron: line) are shown below the tracks. Regions of each gene loci with elevated H3K9Me3 level and impaired Brg1 binding in KOM cells are highlighted.

    Journal: PLoS Genetics

    Article Title: β-actin regulates a heterochromatin landscape essential for optimal induction of neuronal programs during direct reprograming

    doi: 10.1371/journal.pgen.1007846

    Figure Lengend Snippet: β-actin is required for the regulation of chromatin status and the expression of Zic and Irx genes in MEFs or CiNeurons. (A) The relative expression of Zic amd Irx genes in MEFs and CiNeurons. Data are summary of at least 3 biological replicates from RNA-seq analysis. Error bar: S.E.M. (B) H3K9Me3 and Brg1 ChIP-seq analysis in WTM and KOM cells at Zic and Irx loci. Normalized signal at Zic1 ; Zic2 ; Zic3 ; Zic4 ; Irx1 ; and Irx3 loci were shown. The y-axis data range represents RPKM (Reads Per Kilobase of sequence range per Million mapped reads) per bin. The y-axis of tracks in the same image were set as the same range. Gene body position (exon: box, intron: line) are shown below the tracks. Regions of each gene loci with elevated H3K9Me3 level and impaired Brg1 binding in KOM cells are highlighted.

    Article Snippet: Anti-Brg1 antibody is from Dr. Anki Ӧstlund-Farrants Lab (Department of Molecular Biosciences, University of Stockholm, Sweden).

    Techniques: Expressing, RNA Sequencing Assay, Chromatin Immunoprecipitation, Sequencing, Binding Assay

    β-actin dependent Brg1 chromatin binding and H3K9Me3 changes in MEFs regulate the expression of neurogenic programs in direct reprograming. (A) Schematics of the selection of genes involved in neurogenesis of direct reprograming. 1. The genes differentially expressed WTM and KOM were removed. 2. After the filter 1, the remaining genes that are up-regulated by at least 2 fold in β-actin +/+ WT background during direct reprograming (up-regulated in WTN in comparison to WTM) were further selected. 3. Followed by the filter 2, the genes were further filtered as follows: genes up-regulated in KON in comparison to WTN (3a), genes down-regulated in KON (3b) or genes that are not differentially expressed (3c). The final lists of filtered gene in 3a, 3b and 3c were selected respectively for the downstream analysis of H3K9Me3 and Brg1 binding profiles. (B) Average H3K9Me3 Chip-seq signal within ± 5kb of TSS of genes in gene lists of 3a, 3b, and 3c between WTM and KOM. (C) The difference of H3K9Me3 Chip-seq signal in each10 bp bin within ± 5kb of TSS between KOM and WTM was calculated. Violin diagram shows the distribution of the relative ChIP signal difference (RPKM) at each 10 bp bin between KOM and WTM. One-way ANOVA with Tukey’s post hoc test: *** p

    Journal: PLoS Genetics

    Article Title: β-actin regulates a heterochromatin landscape essential for optimal induction of neuronal programs during direct reprograming

    doi: 10.1371/journal.pgen.1007846

    Figure Lengend Snippet: β-actin dependent Brg1 chromatin binding and H3K9Me3 changes in MEFs regulate the expression of neurogenic programs in direct reprograming. (A) Schematics of the selection of genes involved in neurogenesis of direct reprograming. 1. The genes differentially expressed WTM and KOM were removed. 2. After the filter 1, the remaining genes that are up-regulated by at least 2 fold in β-actin +/+ WT background during direct reprograming (up-regulated in WTN in comparison to WTM) were further selected. 3. Followed by the filter 2, the genes were further filtered as follows: genes up-regulated in KON in comparison to WTN (3a), genes down-regulated in KON (3b) or genes that are not differentially expressed (3c). The final lists of filtered gene in 3a, 3b and 3c were selected respectively for the downstream analysis of H3K9Me3 and Brg1 binding profiles. (B) Average H3K9Me3 Chip-seq signal within ± 5kb of TSS of genes in gene lists of 3a, 3b, and 3c between WTM and KOM. (C) The difference of H3K9Me3 Chip-seq signal in each10 bp bin within ± 5kb of TSS between KOM and WTM was calculated. Violin diagram shows the distribution of the relative ChIP signal difference (RPKM) at each 10 bp bin between KOM and WTM. One-way ANOVA with Tukey’s post hoc test: *** p

    Article Snippet: Anti-Brg1 antibody is from Dr. Anki Ӧstlund-Farrants Lab (Department of Molecular Biosciences, University of Stockholm, Sweden).

    Techniques: Binding Assay, Expressing, Selection, Chromatin Immunoprecipitation

    BRG1 protein expression in NSCs (a) Western immunoblot shows a single BRG1 band at ~238KD detected in the nuclear extract of neurosphere cultures but not in the cytoplasmic extract. (b) BRG1 protein levels in neurospheres after 0mg/dl, 120mg/dl and 320mg/dl ethanol treatment. Lower panel shows the corresponding immunoblot when probed for β-Actin as a loading control. (c) Bar graph, depicting the quantification of the western blot, shows that ethanol did not alter BRG1 protein expression, consistent with the lack of effect on BRG1 mRNA expression. The vertical axis shows the ratio of the band density of BRG1 to the ratio of the band density of β-Actin. Error bars indicates standard error of the mean. (d) Western blot analysis of BRG1 immunoprecipitation in cytoplasmic and nuclear cellular fractions probed with anti-BRG1 antibody. BRG1 is specifically precipitated with anti-BRG1 antibody (blue text), but not with an isotype-specific IgG control antibody (red text). Efficiency of anti-BRG1 immunoprecipitation is indicated by the relative depletion of BRG1 from the nuclear supernatant and enrichment in the immuno-precipitate (IP).

    Journal: Alcohol (Fayetteville, N.Y.)

    Article Title: The BAF (BRG1/BRM-Associated Factor) Chromatin-Remodeling Complex Exhibits Ethanol Sensitivity in Fetal Neural Progenitor Cells and Regulates Transcription at the Mir-9-2 Encoding Gene Locus

    doi: 10.1016/j.alcohol.2017.01.003

    Figure Lengend Snippet: BRG1 protein expression in NSCs (a) Western immunoblot shows a single BRG1 band at ~238KD detected in the nuclear extract of neurosphere cultures but not in the cytoplasmic extract. (b) BRG1 protein levels in neurospheres after 0mg/dl, 120mg/dl and 320mg/dl ethanol treatment. Lower panel shows the corresponding immunoblot when probed for β-Actin as a loading control. (c) Bar graph, depicting the quantification of the western blot, shows that ethanol did not alter BRG1 protein expression, consistent with the lack of effect on BRG1 mRNA expression. The vertical axis shows the ratio of the band density of BRG1 to the ratio of the band density of β-Actin. Error bars indicates standard error of the mean. (d) Western blot analysis of BRG1 immunoprecipitation in cytoplasmic and nuclear cellular fractions probed with anti-BRG1 antibody. BRG1 is specifically precipitated with anti-BRG1 antibody (blue text), but not with an isotype-specific IgG control antibody (red text). Efficiency of anti-BRG1 immunoprecipitation is indicated by the relative depletion of BRG1 from the nuclear supernatant and enrichment in the immuno-precipitate (IP).

    Article Snippet: The sonicated nuclear lysate was then treated with either anti-BRG1 antibody or goat anti-rabbit IgG (Thermo Fisher Scientific) and incubated overnight.

    Techniques: Expressing, Western Blot, Immunoprecipitation

    ChIP analysis indicates that ethanol exposure increases BRG1 binding to DNAse I-resistant/CpG island containing region 3 and the pre-miR-9-2 coding exon Bar graph shows the effect of ethanol on BRG1-association with regions 1 to 5 and the pre-miR-9-2 exon-coding region of the primary (pri)-miR-9-2 coding locus. Primers for a gene desert on chromosome 6 (not predicted to bind any transcription factors) shows the specificity of the immuno-precipitation. Vertical axis shows the fold change of the specific associated DNA region to BRG1 in ethanol treated group relative to control group. Error bars indicate standard error of the mean.

    Journal: Alcohol (Fayetteville, N.Y.)

    Article Title: The BAF (BRG1/BRM-Associated Factor) Chromatin-Remodeling Complex Exhibits Ethanol Sensitivity in Fetal Neural Progenitor Cells and Regulates Transcription at the Mir-9-2 Encoding Gene Locus

    doi: 10.1016/j.alcohol.2017.01.003

    Figure Lengend Snippet: ChIP analysis indicates that ethanol exposure increases BRG1 binding to DNAse I-resistant/CpG island containing region 3 and the pre-miR-9-2 coding exon Bar graph shows the effect of ethanol on BRG1-association with regions 1 to 5 and the pre-miR-9-2 exon-coding region of the primary (pri)-miR-9-2 coding locus. Primers for a gene desert on chromosome 6 (not predicted to bind any transcription factors) shows the specificity of the immuno-precipitation. Vertical axis shows the fold change of the specific associated DNA region to BRG1 in ethanol treated group relative to control group. Error bars indicate standard error of the mean.

    Article Snippet: The sonicated nuclear lysate was then treated with either anti-BRG1 antibody or goat anti-rabbit IgG (Thermo Fisher Scientific) and incubated overnight.

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation

    BRG1-containing complexes associate with both DNAse I-hypersensitive and insensitive sites on the miR-9-2 gene locus (a) Positional representation of POU5F1/Oct4, c-myc and REST transcription regulatory factor binding sites along the human pre-miR-9-2 gene locus identified with the UCSC genome browser ENCODE analysis hub track. Colored circles indicate locations for primer pairs for pri-miR-9-2 regions 1,2,3,4 and 5 and the pre-miR-9-2 coding region. (b) qPCR results of BRG1-ChIP from neurospheres. Primers were used to amplify 6 distinct positions along the pri-miR-9-2 gene locus and a genetically sparse region on mouse chromosome 6. Vertical axis shows fold change relative to an IgG pulldown control. Error bars indicate standard error of the mean. (c) qPCR of neurosphere DNA following digestion with 20, 60 and 120U of DNAse I at the 6 positions along the pri-miR-9-2 gene locus. The vertical axis shows fold amplification relative to DNAse I-untreated neurosphere DNA.

    Journal: Alcohol (Fayetteville, N.Y.)

    Article Title: The BAF (BRG1/BRM-Associated Factor) Chromatin-Remodeling Complex Exhibits Ethanol Sensitivity in Fetal Neural Progenitor Cells and Regulates Transcription at the Mir-9-2 Encoding Gene Locus

    doi: 10.1016/j.alcohol.2017.01.003

    Figure Lengend Snippet: BRG1-containing complexes associate with both DNAse I-hypersensitive and insensitive sites on the miR-9-2 gene locus (a) Positional representation of POU5F1/Oct4, c-myc and REST transcription regulatory factor binding sites along the human pre-miR-9-2 gene locus identified with the UCSC genome browser ENCODE analysis hub track. Colored circles indicate locations for primer pairs for pri-miR-9-2 regions 1,2,3,4 and 5 and the pre-miR-9-2 coding region. (b) qPCR results of BRG1-ChIP from neurospheres. Primers were used to amplify 6 distinct positions along the pri-miR-9-2 gene locus and a genetically sparse region on mouse chromosome 6. Vertical axis shows fold change relative to an IgG pulldown control. Error bars indicate standard error of the mean. (c) qPCR of neurosphere DNA following digestion with 20, 60 and 120U of DNAse I at the 6 positions along the pri-miR-9-2 gene locus. The vertical axis shows fold amplification relative to DNAse I-untreated neurosphere DNA.

    Article Snippet: The sonicated nuclear lysate was then treated with either anti-BRG1 antibody or goat anti-rabbit IgG (Thermo Fisher Scientific) and incubated overnight.

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