smarca4 gene Search Results


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  • 87
    Thermo Fisher gene exp smarca4 hs00231324 m1
    KEGG pathway analysis of <t>BRG1</t> co-expressed genes. a Positively co-expressed genes. b Negatively co-expressed genes. Enrichment number means the percentage of BRG1 co-expressed genes in the pathway.
    Gene Exp Smarca4 Hs00231324 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore brg1 proteins
    <t>Brg1</t> is phosphorylated in vitro by CK2. A, schematic representation of the location of the putative sites for CK2 phosphorylation identified by NetphosK ( 9 , 20 ). B, representative autoradiograms of in vitro CK2-treated WT-, SA-, and SE-Brg1 ( top ). A representative Western blot detecting Brg1 is shown for comparison ( bottom ). EV, empty vector. C, Brg1 labeling was quantified by normalizing autoradiography signals to the Western blotting for Brg1. Data represent the average of three independent experiments ± S.D.; *, p
    Brg1 Proteins, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher gene exp smarca4 hs00946396 m1
    <t>BRG1</t> reduction causes specific changes in gene expression
    Gene Exp Smarca4 Hs00946396 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gene exp smarca4 mm01151948 m1
    <t>BRG1</t> knockdown in F9 cells using SMARTpool siRNA and OCT4 target genes. (A) SiRNA-mediated knockdown of Brg1 in F9 cells was achieved with about 90% efficiency. (B) Levels of Oct4 were increased, Sox2 levels were decreased, whereas Nanog levels remained unchanged in F9 cells at 48 h posttransfection. (C) OCT4 target genes Oct11 and Fgf4 were upregulated in BRG1-knockdown ES cells at 48 h posttransfection. (D) Oct11 levels were also increased in BRG1-knockdown F9 cells at 72 h posttransfection.
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    PreventionGenetics smarca4 gene
    ( A ) OncoScan Nexus Copy Number 7.5 (Nexus) view showing a diploid 46,XX array with focal abnormalities in Chromosomes 17p and 19p (arrows). ( B ) Integrative Genomics Viewer (IGV) 2.3 view of TP53 c.594_611del18 in-frame deletion of 18 nt at 25.9% variant allele fraction (VAF) ( left ). Nexus view showing mosaic copy-neutral loss of heterozygosity (CN-LOH) of Chromosome 17p13.3-p11.2 including the TP53 locus ( right ; log 2 ratio and B-allele frequency view). ( C ) IGV 2.3 view of orthogonal targeted next-generation sequencing confirmation of <t>SMARCA4</t> c.1141C > T single-nucleotide variant at 88% VAF ( left ). Nexus view showing CN-LOH of Chromosome 19p13.3-19p13.2 including the SMARCA4 locus ( right ; log 2 ratio and allele peak view).
    Smarca4 Gene, supplied by PreventionGenetics, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ECM Biosciences smarca4
    ( A ) Compartment profiles (the first principal components) of shSCRAM and sh <t>SMARCA4</t> data for Chr 2. The A-type (open) compartments are shown in black, and the B-type (closed) compartments are shown in gray. The same color scheme was used for the gene density
    Smarca4, supplied by ECM Biosciences, used in various techniques. Bioz Stars score: 91/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology brg1
    <t>BRG1</t> and NF-κB form a DNA binding complex in myelinating cells (A) P2 or P5 mouse sciatic nerve lysates were analyzed for protein binding to DNA using a consensus sequence for NF-κB in an electrophoretic mobility shift assay (EMSA). To identify the proteins in the complex bound to the NF-κB consensus sites, specific antibodies (ab) to BRG1 (2 different antibodies were used, as indicated), p65 or a control antibody were added to the binding reaction. The complex and unbound DNA was then separated by gel electrophoresis. Excess unlabeled NF- κB consensus DNA (cold) was added as a control (n=3). Note the reduction in the binding complex caused by either BRG1 antibody and the antibody to p65. Similar EMSAs were performed on nerve lysates using consensus sequences for EGR2/Krox20 and Oct6, as indicated. (B) EMSA analysis using an NF-κB probe was performed on lysates from DRG/Schwann cell co-cultures either prior to stimulation with ascorbic acid (AA), to induce myelination, or after 6 days of AA treatment, in the presence or absence of antibodies to BRG1 or control (n=3). (C) HEK 293 cells were treated with TNFα for 1 hr to stimulate NF-κB, lysed and the lysates subjected to an EMSA using an NF-κB consensus sequence. In contrast to experiments in myelinating cells, addition of antibodies to BRG1 had no effect on the protein-DNA complex. (D) P2 rat sciatic nerve lysate was analyzed by EMSA using a NF-κB probe. Supershift analysis was used to identify DNA bound proteins. Addition of antibodies to BRM or control had no effect on protein-DNA binding. Unlabeled probe (cold) was added as a control.
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    Santa Cruz Biotechnology brm swi2 related gene 1 brg1
    <t>BRG1</t> and NF-κB form a DNA binding complex in myelinating cells (A) P2 or P5 mouse sciatic nerve lysates were analyzed for protein binding to DNA using a consensus sequence for NF-κB in an electrophoretic mobility shift assay (EMSA). To identify the proteins in the complex bound to the NF-κB consensus sites, specific antibodies (ab) to BRG1 (2 different antibodies were used, as indicated), p65 or a control antibody were added to the binding reaction. The complex and unbound DNA was then separated by gel electrophoresis. Excess unlabeled NF- κB consensus DNA (cold) was added as a control (n=3). Note the reduction in the binding complex caused by either BRG1 antibody and the antibody to p65. Similar EMSAs were performed on nerve lysates using consensus sequences for EGR2/Krox20 and Oct6, as indicated. (B) EMSA analysis using an NF-κB probe was performed on lysates from DRG/Schwann cell co-cultures either prior to stimulation with ascorbic acid (AA), to induce myelination, or after 6 days of AA treatment, in the presence or absence of antibodies to BRG1 or control (n=3). (C) HEK 293 cells were treated with TNFα for 1 hr to stimulate NF-κB, lysed and the lysates subjected to an EMSA using an NF-κB consensus sequence. In contrast to experiments in myelinating cells, addition of antibodies to BRG1 had no effect on the protein-DNA complex. (D) P2 rat sciatic nerve lysate was analyzed by EMSA using a NF-κB probe. Supershift analysis was used to identify DNA bound proteins. Addition of antibodies to BRM or control had no effect on protein-DNA binding. Unlabeled probe (cold) was added as a control.
    Brm Swi2 Related Gene 1 Brg1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    brg1  (Abcam)
    92
    Abcam brg1
    <t>BRG1</t> controls dynamics of MITF binding. ( A ) ChIP-qPCR of 3HA-MITF in 501Mel-CL8 cells at the indicated loci following transfection with siLuc or siBRG1. The protamine 1 locus (PRM1) was used as a negative control. ( B ) UCSC screenshots illustrating binding of MITF between two BRG1-occupied nucleosomes at selected loci assayed by ChIP-qPCR in panel A . sThe GPR110-1 and GPR110-2 sites assayed in Panel A are indicated in panel B . ( C ) A model for regulatory elements in the melanocyte lineage. Melanocyte lineage enhancers comprise combinations of MITF, SOX10, YY1, and also TFAP2A and ETS1 (not represented for simplicity. Note also that Pol II and the PIC are present at active enhancers where enhancer RNAs are made. For simplicity these are also not represented.) bound to a nucleosome-depleted region. MITF but also these other factors recruit BRG1/PBAF to the nucleosomes flanking the combinations of transcription factors. BRG1/PBAF also occupies the nucleosomes flanking the TSS and a subset of these promoters further comprises a MITF binding site close to the TSS. DOI: http://dx.doi.org/10.7554/eLife.06857.018
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    Thermo Fisher smarca4 rnai
    <t>BRG1</t> controls dynamics of MITF binding. ( A ) ChIP-qPCR of 3HA-MITF in 501Mel-CL8 cells at the indicated loci following transfection with siLuc or siBRG1. The protamine 1 locus (PRM1) was used as a negative control. ( B ) UCSC screenshots illustrating binding of MITF between two BRG1-occupied nucleosomes at selected loci assayed by ChIP-qPCR in panel A . sThe GPR110-1 and GPR110-2 sites assayed in Panel A are indicated in panel B . ( C ) A model for regulatory elements in the melanocyte lineage. Melanocyte lineage enhancers comprise combinations of MITF, SOX10, YY1, and also TFAP2A and ETS1 (not represented for simplicity. Note also that Pol II and the PIC are present at active enhancers where enhancer RNAs are made. For simplicity these are also not represented.) bound to a nucleosome-depleted region. MITF but also these other factors recruit BRG1/PBAF to the nucleosomes flanking the combinations of transcription factors. BRG1/PBAF also occupies the nucleosomes flanking the TSS and a subset of these promoters further comprises a MITF binding site close to the TSS. DOI: http://dx.doi.org/10.7554/eLife.06857.018
    Smarca4 Rnai, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher brg1
    <t>BRG1</t> 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.
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    Epitomics brahma related gene 1
    <t>BRG1</t> 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.
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    4Gene member 4 gene
    <t>BRG1</t> 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.
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    Millipore lys9 dimethylated histone h3 brm related gene 1
    <t>BRG1</t> 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.
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    Genechem hairpin rna against baf155
    <t>BRG1</t> 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.
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    Genechem hairpin rna targeting baf155
    <t>BRG1</t> 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.
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    Image Search Results


    KEGG pathway analysis of BRG1 co-expressed genes. a Positively co-expressed genes. b Negatively co-expressed genes. Enrichment number means the percentage of BRG1 co-expressed genes in the pathway.

    Journal: Cell Death & Disease

    Article Title: Oncogene-dependent function of BRG1 in hepatocarcinogenesis

    doi: 10.1038/s41419-020-2289-3

    Figure Lengend Snippet: KEGG pathway analysis of BRG1 co-expressed genes. a Positively co-expressed genes. b Negatively co-expressed genes. Enrichment number means the percentage of BRG1 co-expressed genes in the pathway.

    Article Snippet: To determine the levels of BRG1 in our HCC collection, Gene Expression Assays for human BRG1/SMARCA4 (ID# Hs00231324_m1) and β-Actin (ID #4333762 T) genes were purchased from Applied Biosystems (Foster City, CA, USA).

    Techniques:

    Brg1 inactivation prevents hepatocellular carcinoma (HCC) formation in c-MYC mice. a Study design. b Survival analysis of Brg1 f/f mice bearing c-MYC/pCMV and c-MYC /CRE tumors using the Kaplan–Meier survival method. c Liver body weight ratio of Brg1 f/f c-MYC/pCMV ( n = 9) and c-MYC /CRE mice ( n = 11). d Gross image, H E staining, Brg1, and Ki67 staining of Brg1 f/f c-MYC/pCMV and c-MYC /CRE mice.

    Journal: Cell Death & Disease

    Article Title: Oncogene-dependent function of BRG1 in hepatocarcinogenesis

    doi: 10.1038/s41419-020-2289-3

    Figure Lengend Snippet: Brg1 inactivation prevents hepatocellular carcinoma (HCC) formation in c-MYC mice. a Study design. b Survival analysis of Brg1 f/f mice bearing c-MYC/pCMV and c-MYC /CRE tumors using the Kaplan–Meier survival method. c Liver body weight ratio of Brg1 f/f c-MYC/pCMV ( n = 9) and c-MYC /CRE mice ( n = 11). d Gross image, H E staining, Brg1, and Ki67 staining of Brg1 f/f c-MYC/pCMV and c-MYC /CRE mice.

    Article Snippet: To determine the levels of BRG1 in our HCC collection, Gene Expression Assays for human BRG1/SMARCA4 (ID# Hs00231324_m1) and β-Actin (ID #4333762 T) genes were purchased from Applied Biosystems (Foster City, CA, USA).

    Techniques: Mouse Assay, Staining

    Ablation of Brg1 in mouse hepatocytes does not affect liver homeostasis in adult mice. a Study design. b Protein expression of Brg1 in the liver of Brg1 flf AAV-Cre and of Brg1 flf AAV-null mice analyzed by western blot analysis. c Gross image, H E staining, Ki67, and Brg1 staining of Brg1 flf AAV-Cre and of Brg1 flf AAV-null mice. d Liver body weight ratio of Brg1 flf AAV-Cre ( n = 6) and Brg1 flf AAV-null mice ( n = 7).

    Journal: Cell Death & Disease

    Article Title: Oncogene-dependent function of BRG1 in hepatocarcinogenesis

    doi: 10.1038/s41419-020-2289-3

    Figure Lengend Snippet: Ablation of Brg1 in mouse hepatocytes does not affect liver homeostasis in adult mice. a Study design. b Protein expression of Brg1 in the liver of Brg1 flf AAV-Cre and of Brg1 flf AAV-null mice analyzed by western blot analysis. c Gross image, H E staining, Ki67, and Brg1 staining of Brg1 flf AAV-Cre and of Brg1 flf AAV-null mice. d Liver body weight ratio of Brg1 flf AAV-Cre ( n = 6) and Brg1 flf AAV-null mice ( n = 7).

    Article Snippet: To determine the levels of BRG1 in our HCC collection, Gene Expression Assays for human BRG1/SMARCA4 (ID# Hs00231324_m1) and β-Actin (ID #4333762 T) genes were purchased from Applied Biosystems (Foster City, CA, USA).

    Techniques: Mouse Assay, Expressing, Western Blot, Staining

    BRG1 expression, mutation, and survival analysis in human data sets. a Scatter-bar plot of BRG1 mRNA expression in TCGA LIHC data set. b Scatter-bar plot of BRG1 mRNA expression in Fudan data set. c Kaplan–Meier survival plot from UALCAN using TCGA LIHC data set. d Heatmap of BRG1 mRNA expression in TCGA LIHC data set with multiple mutation status of well-known oncogenes in HCC. ST surrounding tissue, HCC hepatocellular carcinoma; **** p

    Journal: Cell Death & Disease

    Article Title: Oncogene-dependent function of BRG1 in hepatocarcinogenesis

    doi: 10.1038/s41419-020-2289-3

    Figure Lengend Snippet: BRG1 expression, mutation, and survival analysis in human data sets. a Scatter-bar plot of BRG1 mRNA expression in TCGA LIHC data set. b Scatter-bar plot of BRG1 mRNA expression in Fudan data set. c Kaplan–Meier survival plot from UALCAN using TCGA LIHC data set. d Heatmap of BRG1 mRNA expression in TCGA LIHC data set with multiple mutation status of well-known oncogenes in HCC. ST surrounding tissue, HCC hepatocellular carcinoma; **** p

    Article Snippet: To determine the levels of BRG1 in our HCC collection, Gene Expression Assays for human BRG1/SMARCA4 (ID# Hs00231324_m1) and β-Actin (ID #4333762 T) genes were purchased from Applied Biosystems (Foster City, CA, USA).

    Techniques: Expressing, Mutagenesis

    Brg1 deletion cooperates with c-MET and NRAS V12 to induce liver tumor formation in mice. a Study design. b Survival analysis of Brg1 f/f mice injected CRE plasmid and c-MET (Brg1 −/− /c-MET) or NRAS V12 (Brg1 −/− / NRAS V12 ). c Liver body weight ratio of Brg1 −/− /c-MET ( n = 21) and Brg1 −/− / NRAS V12 mice ( n = 9). d Gross image, H E staining, Brg1, and Ki67 staining of Brg1 −/− /c-MET and Brg1 −/− / NRAS V12 mice.

    Journal: Cell Death & Disease

    Article Title: Oncogene-dependent function of BRG1 in hepatocarcinogenesis

    doi: 10.1038/s41419-020-2289-3

    Figure Lengend Snippet: Brg1 deletion cooperates with c-MET and NRAS V12 to induce liver tumor formation in mice. a Study design. b Survival analysis of Brg1 f/f mice injected CRE plasmid and c-MET (Brg1 −/− /c-MET) or NRAS V12 (Brg1 −/− / NRAS V12 ). c Liver body weight ratio of Brg1 −/− /c-MET ( n = 21) and Brg1 −/− / NRAS V12 mice ( n = 9). d Gross image, H E staining, Brg1, and Ki67 staining of Brg1 −/− /c-MET and Brg1 −/− / NRAS V12 mice.

    Article Snippet: To determine the levels of BRG1 in our HCC collection, Gene Expression Assays for human BRG1/SMARCA4 (ID# Hs00231324_m1) and β-Actin (ID #4333762 T) genes were purchased from Applied Biosystems (Foster City, CA, USA).

    Techniques: Mouse Assay, Injection, Plasmid Preparation, Staining

    Brg1 is phosphorylated in vitro by CK2. A, schematic representation of the location of the putative sites for CK2 phosphorylation identified by NetphosK ( 9 , 20 ). B, representative autoradiograms of in vitro CK2-treated WT-, SA-, and SE-Brg1 ( top ). A representative Western blot detecting Brg1 is shown for comparison ( bottom ). EV, empty vector. C, Brg1 labeling was quantified by normalizing autoradiography signals to the Western blotting for Brg1. Data represent the average of three independent experiments ± S.D.; *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Casein kinase 2-mediated phosphorylation of Brahma-related gene 1 controls myoblast proliferation and contributes to SWI/SNF complex composition

    doi: 10.1074/jbc.M117.799676

    Figure Lengend Snippet: Brg1 is phosphorylated in vitro by CK2. A, schematic representation of the location of the putative sites for CK2 phosphorylation identified by NetphosK ( 9 , 20 ). B, representative autoradiograms of in vitro CK2-treated WT-, SA-, and SE-Brg1 ( top ). A representative Western blot detecting Brg1 is shown for comparison ( bottom ). EV, empty vector. C, Brg1 labeling was quantified by normalizing autoradiography signals to the Western blotting for Brg1. Data represent the average of three independent experiments ± S.D.; *, p

    Article Snippet: The resulting Brg1 proteins were immunoprecipitated using a polyclonal rabbit antisera against Brg1 ( ) and PureProteomeTM protein A/G mix magnetic beads (Millipore).

    Techniques: In Vitro, Western Blot, Plasmid Preparation, Labeling, Autoradiography

    Inhibition of CK2 affects CK2-mediated in vitro phosphorylation of Brg1 and primary myoblast proliferation. A, dose-dependent inhibition of CK2-mediated in vitro phosphorylation of Brg1 by TBB. Representative autoradiograms ( top ) and Western blots ( WB ) confirming the expression of Brg1 ( bottom ) are shown. B, quantification of phosphorylation changes presented as the mean ± S.D. from four independent experiments *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Casein kinase 2-mediated phosphorylation of Brahma-related gene 1 controls myoblast proliferation and contributes to SWI/SNF complex composition

    doi: 10.1074/jbc.M117.799676

    Figure Lengend Snippet: Inhibition of CK2 affects CK2-mediated in vitro phosphorylation of Brg1 and primary myoblast proliferation. A, dose-dependent inhibition of CK2-mediated in vitro phosphorylation of Brg1 by TBB. Representative autoradiograms ( top ) and Western blots ( WB ) confirming the expression of Brg1 ( bottom ) are shown. B, quantification of phosphorylation changes presented as the mean ± S.D. from four independent experiments *, p

    Article Snippet: The resulting Brg1 proteins were immunoprecipitated using a polyclonal rabbit antisera against Brg1 ( ) and PureProteomeTM protein A/G mix magnetic beads (Millipore).

    Techniques: Inhibition, In Vitro, Western Blot, Expressing

    Phosphomimetic Brg1 mutant does not bind to or remodel chromatin at the Pax7 promotor. A, mRNA expression levels of Pax7 in the indicated primary myoblasts. Values for the C57Bl/6 primary myoblasts were set at 1. B and C, quantification of ChIP assays measuring binding of Brg1 to the Pax7 promoter ( B ) or the IgH enhancer ( C ) in each of the indicated primary myoblasts. D, schematic representation of the location of the PvuII sites and the primer sets used for REAA. E and F, quantification of REAAs performed on the indicated primary myoblasts using primer sets 1 and 2 ( E ) or primer sets 3 and 4 ( F ). The accessibility at the PvuII site assessed by primer set 1 in the WT-Brg1-expressing primary myoblasts was set at 1. All data represent the average of three independent experiments, each assayed in triplicate ± S.D.; *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Casein kinase 2-mediated phosphorylation of Brahma-related gene 1 controls myoblast proliferation and contributes to SWI/SNF complex composition

    doi: 10.1074/jbc.M117.799676

    Figure Lengend Snippet: Phosphomimetic Brg1 mutant does not bind to or remodel chromatin at the Pax7 promotor. A, mRNA expression levels of Pax7 in the indicated primary myoblasts. Values for the C57Bl/6 primary myoblasts were set at 1. B and C, quantification of ChIP assays measuring binding of Brg1 to the Pax7 promoter ( B ) or the IgH enhancer ( C ) in each of the indicated primary myoblasts. D, schematic representation of the location of the PvuII sites and the primer sets used for REAA. E and F, quantification of REAAs performed on the indicated primary myoblasts using primer sets 1 and 2 ( E ) or primer sets 3 and 4 ( F ). The accessibility at the PvuII site assessed by primer set 1 in the WT-Brg1-expressing primary myoblasts was set at 1. All data represent the average of three independent experiments, each assayed in triplicate ± S.D.; *, p

    Article Snippet: The resulting Brg1 proteins were immunoprecipitated using a polyclonal rabbit antisera against Brg1 ( ) and PureProteomeTM protein A/G mix magnetic beads (Millipore).

    Techniques: Mutagenesis, Expressing, Chromatin Immunoprecipitation, Binding Assay

    Phosphomimetic mutations in Brg1 inhibit proliferation and reduce viability of primary myoblasts. A, proliferation assay of Brg1-deficient (Brg1 c/c) cells transduced with WT-, SA-, or SE-Brg1. Data represent the average of three independent experiments ± S.D. B, representative Western blots ( WB ) showing the expression of Brg1, Pax7, activated caspase 3, and PI3K as a loading control. C, representative confocal microscopy images showing nuclear localization of WT-, SA-, and SE-Brg1 expressed in proliferating primary myoblasts. D, representative confocal microscopy images of primary myoblasts transduced with the WT-, SA-, and SE-Brg1 mutants that were differentiated for 48 h and immunostained for Brg1 and MyoD. E, representative light microscopy images of the phenotypes observed when myoblasts expressing the indicated Brg1 protein were differentiated for 48 h; cells were immunostained using an anti-myosin heavy chain II ( MHCII ) antibody.

    Journal: The Journal of Biological Chemistry

    Article Title: Casein kinase 2-mediated phosphorylation of Brahma-related gene 1 controls myoblast proliferation and contributes to SWI/SNF complex composition

    doi: 10.1074/jbc.M117.799676

    Figure Lengend Snippet: Phosphomimetic mutations in Brg1 inhibit proliferation and reduce viability of primary myoblasts. A, proliferation assay of Brg1-deficient (Brg1 c/c) cells transduced with WT-, SA-, or SE-Brg1. Data represent the average of three independent experiments ± S.D. B, representative Western blots ( WB ) showing the expression of Brg1, Pax7, activated caspase 3, and PI3K as a loading control. C, representative confocal microscopy images showing nuclear localization of WT-, SA-, and SE-Brg1 expressed in proliferating primary myoblasts. D, representative confocal microscopy images of primary myoblasts transduced with the WT-, SA-, and SE-Brg1 mutants that were differentiated for 48 h and immunostained for Brg1 and MyoD. E, representative light microscopy images of the phenotypes observed when myoblasts expressing the indicated Brg1 protein were differentiated for 48 h; cells were immunostained using an anti-myosin heavy chain II ( MHCII ) antibody.

    Article Snippet: The resulting Brg1 proteins were immunoprecipitated using a polyclonal rabbit antisera against Brg1 ( ) and PureProteomeTM protein A/G mix magnetic beads (Millipore).

    Techniques: Proliferation Assay, Transduction, Western Blot, Expressing, Confocal Microscopy, Light Microscopy

    Brg1 and CK2 interact in primary myoblasts. Gene ( A ) and protein ( B ) expression of CK2α, CK2α′, and CK2β do not change over the course of differentiation of primary myoblasts. Expression in proliferating ( P ) primary myoblasts was set at 1. Data represent the average of three independent experiments, each assayed in triplicate ± S.D. Observed differences were not statistically significant. MHC II levels are shown as a differentiation control, and PI3K levels are shown as a loading control for the Western blots ( WB ). C, Brg1 and CK2 interact in proliferating primary myoblasts. A representative Western blot of the reciprocal immunoprecipitations ( IP )of Brg1 and CK2α is shown. Pulldown with IgG was used as a negative control.

    Journal: The Journal of Biological Chemistry

    Article Title: Casein kinase 2-mediated phosphorylation of Brahma-related gene 1 controls myoblast proliferation and contributes to SWI/SNF complex composition

    doi: 10.1074/jbc.M117.799676

    Figure Lengend Snippet: Brg1 and CK2 interact in primary myoblasts. Gene ( A ) and protein ( B ) expression of CK2α, CK2α′, and CK2β do not change over the course of differentiation of primary myoblasts. Expression in proliferating ( P ) primary myoblasts was set at 1. Data represent the average of three independent experiments, each assayed in triplicate ± S.D. Observed differences were not statistically significant. MHC II levels are shown as a differentiation control, and PI3K levels are shown as a loading control for the Western blots ( WB ). C, Brg1 and CK2 interact in proliferating primary myoblasts. A representative Western blot of the reciprocal immunoprecipitations ( IP )of Brg1 and CK2α is shown. Pulldown with IgG was used as a negative control.

    Article Snippet: The resulting Brg1 proteins were immunoprecipitated using a polyclonal rabbit antisera against Brg1 ( ) and PureProteomeTM protein A/G mix magnetic beads (Millipore).

    Techniques: Expressing, Western Blot, Negative Control

    Inhibition of CK2 or expression of phosphomimetic or non-phosphorylatable Brg1 mutations determines interaction with Baf170 or Baf155. A, representative Western blots ( WB ) showing 12% of the input for the subsequent immunoprecipitation ( IP ) experiments, which documents expression of Baf170 and Baf155 in the indicated proliferating primary myoblasts. Bands were quantified using ImageJ. Brg1 expression was normalized to PI3K expression, and the value of expression in the C57Bl/6 myoblasts was set at 1. Relative expression levels are indicated below each band. B, representative profiles of Baf170 and Baf155 immunoprecipitations of Brg1 from the indicated primary myoblasts. C and D, ChIP experiments showing Baf170 recruitment to the Pax7 promoter ( C ) or to the IgH enhancer ( D ) when endogenous or WT-Brg1 is expressed. E and F, ChIP experiments showing Baf155 recruitment to the Pax7 promoter ( E ) or to the IgH enhancer ( F ) when the non-phosphorylatable Brg1 mutant is expressed. Data in C–F represent the average of three independent experiments, each assayed in triplicate ± S.D.; *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Casein kinase 2-mediated phosphorylation of Brahma-related gene 1 controls myoblast proliferation and contributes to SWI/SNF complex composition

    doi: 10.1074/jbc.M117.799676

    Figure Lengend Snippet: Inhibition of CK2 or expression of phosphomimetic or non-phosphorylatable Brg1 mutations determines interaction with Baf170 or Baf155. A, representative Western blots ( WB ) showing 12% of the input for the subsequent immunoprecipitation ( IP ) experiments, which documents expression of Baf170 and Baf155 in the indicated proliferating primary myoblasts. Bands were quantified using ImageJ. Brg1 expression was normalized to PI3K expression, and the value of expression in the C57Bl/6 myoblasts was set at 1. Relative expression levels are indicated below each band. B, representative profiles of Baf170 and Baf155 immunoprecipitations of Brg1 from the indicated primary myoblasts. C and D, ChIP experiments showing Baf170 recruitment to the Pax7 promoter ( C ) or to the IgH enhancer ( D ) when endogenous or WT-Brg1 is expressed. E and F, ChIP experiments showing Baf155 recruitment to the Pax7 promoter ( E ) or to the IgH enhancer ( F ) when the non-phosphorylatable Brg1 mutant is expressed. Data in C–F represent the average of three independent experiments, each assayed in triplicate ± S.D.; *, p

    Article Snippet: The resulting Brg1 proteins were immunoprecipitated using a polyclonal rabbit antisera against Brg1 ( ) and PureProteomeTM protein A/G mix magnetic beads (Millipore).

    Techniques: Inhibition, Expressing, Western Blot, Immunoprecipitation, Chromatin Immunoprecipitation, Mutagenesis

    Phosphomimetic and non-phosphorylatable mutations in Brg1 or inhibition of CK2 affects intranuclear mobility and association with chromatin and the nuclear matrix. A, time course of representative confocal microscopy images from FRAP experiments performed on primary myoblasts expressing WT-, SA-, or SE-Brg1. B, quantification of the fluorescence recovery of WT-, SA-, and SE-Brg1 mutants. Data represent the average values from five independently examined cells ± S.E. n.d., not detected. C, representative Western blottings ( WB ) of Brg1 in the indicated fractions derived from proliferating primary myoblasts. D, representative Western blottings from C57Bl/6 myoblasts for marker proteins to demonstrate the purity of the fractions used in C. E, representative Western blots of Brg1 and marker proteins present in the indicated fractions from primary myoblasts treated with TBB.

    Journal: The Journal of Biological Chemistry

    Article Title: Casein kinase 2-mediated phosphorylation of Brahma-related gene 1 controls myoblast proliferation and contributes to SWI/SNF complex composition

    doi: 10.1074/jbc.M117.799676

    Figure Lengend Snippet: Phosphomimetic and non-phosphorylatable mutations in Brg1 or inhibition of CK2 affects intranuclear mobility and association with chromatin and the nuclear matrix. A, time course of representative confocal microscopy images from FRAP experiments performed on primary myoblasts expressing WT-, SA-, or SE-Brg1. B, quantification of the fluorescence recovery of WT-, SA-, and SE-Brg1 mutants. Data represent the average values from five independently examined cells ± S.E. n.d., not detected. C, representative Western blottings ( WB ) of Brg1 in the indicated fractions derived from proliferating primary myoblasts. D, representative Western blottings from C57Bl/6 myoblasts for marker proteins to demonstrate the purity of the fractions used in C. E, representative Western blots of Brg1 and marker proteins present in the indicated fractions from primary myoblasts treated with TBB.

    Article Snippet: The resulting Brg1 proteins were immunoprecipitated using a polyclonal rabbit antisera against Brg1 ( ) and PureProteomeTM protein A/G mix magnetic beads (Millipore).

    Techniques: Inhibition, Confocal Microscopy, Expressing, Fluorescence, Western Blot, Derivative Assay, Marker

    BRG1 reduction causes specific changes in gene expression

    Journal: Cancer research

    Article Title: BRG1/SMARCA4 inactivation promotes non-small cell lung cancer aggressiveness by altering chromatin organization

    doi: 10.1158/0008-5472.CAN-14-0061

    Figure Lengend Snippet: BRG1 reduction causes specific changes in gene expression

    Article Snippet: The TaqMan primer and probes setsobtained from Applied Biosystems included BRG1 (Hs00946396_m1), SEM3B (Hs00190328_m1), EHF(Hs00171917_m1), DUSP6 (Hs04329643_s1, IL8 (Hs99999034_m1), SYK (Hs00895377_m1), IFI16(Hs00194261_m1), BATF (Hs00232390_m1) and β-ACTIN (Hs00357333_g1).

    Techniques: Expressing

    Characterization of BRG1 target gene expression in BRG1 knockdown cell lines

    Journal: Cancer research

    Article Title: BRG1/SMARCA4 inactivation promotes non-small cell lung cancer aggressiveness by altering chromatin organization

    doi: 10.1158/0008-5472.CAN-14-0061

    Figure Lengend Snippet: Characterization of BRG1 target gene expression in BRG1 knockdown cell lines

    Article Snippet: The TaqMan primer and probes setsobtained from Applied Biosystems included BRG1 (Hs00946396_m1), SEM3B (Hs00190328_m1), EHF(Hs00171917_m1), DUSP6 (Hs04329643_s1, IL8 (Hs99999034_m1), SYK (Hs00895377_m1), IFI16(Hs00194261_m1), BATF (Hs00232390_m1) and β-ACTIN (Hs00357333_g1).

    Techniques: Expressing

    Gene expression changes in response to BRG1 silencing

    Journal: Cancer research

    Article Title: BRG1/SMARCA4 inactivation promotes non-small cell lung cancer aggressiveness by altering chromatin organization

    doi: 10.1158/0008-5472.CAN-14-0061

    Figure Lengend Snippet: Gene expression changes in response to BRG1 silencing

    Article Snippet: The TaqMan primer and probes setsobtained from Applied Biosystems included BRG1 (Hs00946396_m1), SEM3B (Hs00190328_m1), EHF(Hs00171917_m1), DUSP6 (Hs04329643_s1, IL8 (Hs99999034_m1), SYK (Hs00895377_m1), IFI16(Hs00194261_m1), BATF (Hs00232390_m1) and β-ACTIN (Hs00357333_g1).

    Techniques: Expressing

    BRG1 loss results in variation in nucleosome occupancy and positioning

    Journal: Cancer research

    Article Title: BRG1/SMARCA4 inactivation promotes non-small cell lung cancer aggressiveness by altering chromatin organization

    doi: 10.1158/0008-5472.CAN-14-0061

    Figure Lengend Snippet: BRG1 loss results in variation in nucleosome occupancy and positioning

    Article Snippet: The TaqMan primer and probes setsobtained from Applied Biosystems included BRG1 (Hs00946396_m1), SEM3B (Hs00190328_m1), EHF(Hs00171917_m1), DUSP6 (Hs04329643_s1, IL8 (Hs99999034_m1), SYK (Hs00895377_m1), IFI16(Hs00194261_m1), BATF (Hs00232390_m1) and β-ACTIN (Hs00357333_g1).

    Techniques:

    Expression of BRG1 target genes in lung adenocarcinoma

    Journal: Cancer research

    Article Title: BRG1/SMARCA4 inactivation promotes non-small cell lung cancer aggressiveness by altering chromatin organization

    doi: 10.1158/0008-5472.CAN-14-0061

    Figure Lengend Snippet: Expression of BRG1 target genes in lung adenocarcinoma

    Article Snippet: The TaqMan primer and probes setsobtained from Applied Biosystems included BRG1 (Hs00946396_m1), SEM3B (Hs00190328_m1), EHF(Hs00171917_m1), DUSP6 (Hs04329643_s1, IL8 (Hs99999034_m1), SYK (Hs00895377_m1), IFI16(Hs00194261_m1), BATF (Hs00232390_m1) and β-ACTIN (Hs00357333_g1).

    Techniques: Expressing

    In vitro and in vivo growth properties of BRG1-deficient H358 cell lines

    Journal: Cancer research

    Article Title: BRG1/SMARCA4 inactivation promotes non-small cell lung cancer aggressiveness by altering chromatin organization

    doi: 10.1158/0008-5472.CAN-14-0061

    Figure Lengend Snippet: In vitro and in vivo growth properties of BRG1-deficient H358 cell lines

    Article Snippet: The TaqMan primer and probes setsobtained from Applied Biosystems included BRG1 (Hs00946396_m1), SEM3B (Hs00190328_m1), EHF(Hs00171917_m1), DUSP6 (Hs04329643_s1, IL8 (Hs99999034_m1), SYK (Hs00895377_m1), IFI16(Hs00194261_m1), BATF (Hs00232390_m1) and β-ACTIN (Hs00357333_g1).

    Techniques: In Vitro, In Vivo

    BRG1 knockdown in F9 cells using SMARTpool siRNA and OCT4 target genes. (A) SiRNA-mediated knockdown of Brg1 in F9 cells was achieved with about 90% efficiency. (B) Levels of Oct4 were increased, Sox2 levels were decreased, whereas Nanog levels remained unchanged in F9 cells at 48 h posttransfection. (C) OCT4 target genes Oct11 and Fgf4 were upregulated in BRG1-knockdown ES cells at 48 h posttransfection. (D) Oct11 levels were also increased in BRG1-knockdown F9 cells at 72 h posttransfection.

    Journal: BioResearch Open Access

    Article Title: BRG1 Is Required to Maintain Pluripotency of Murine Embryonic Stem Cells

    doi: 10.1089/biores.2013.0047

    Figure Lengend Snippet: BRG1 knockdown in F9 cells using SMARTpool siRNA and OCT4 target genes. (A) SiRNA-mediated knockdown of Brg1 in F9 cells was achieved with about 90% efficiency. (B) Levels of Oct4 were increased, Sox2 levels were decreased, whereas Nanog levels remained unchanged in F9 cells at 48 h posttransfection. (C) OCT4 target genes Oct11 and Fgf4 were upregulated in BRG1-knockdown ES cells at 48 h posttransfection. (D) Oct11 levels were also increased in BRG1-knockdown F9 cells at 72 h posttransfection.

    Article Snippet: Quantitative reverse-transcription polymerase chain reaction analysis TaqMan Assays-on-Demand was used for the detection of the following genes (TaqMan primer ID): Brg1 (Mm01151948_m1), Oct4 (Mm00658129_gH), Sox2 (Mm00488369_s1), Nanog (Mm02019550_s1), Oct11 (Mm00478284_m1), Fgf4 (Mm00438917_m1), Fgf5 (Mm00438919_m1), Gata4 (Mm00484689_m1), Meox1 (Mm00440285_m1), and T (Mm00436877_m1).

    Techniques:

    Time-course analysis was performed by using shRNA directed against Brg1 . (A) Transfection of shRNA against Brg1 showed up to 90% downregulation of Brg1 in ESD3 cells compared with embryonic stem (ES) cells transfected with control shRNA. (B) Transfection of sh Brg1 led to an increase in Oct4 levels at 24 h posttransfection but a decrease in Oct4 levels at 48 and 72 h posttransfection with sh Brg1 , compared with cells transfected with control shRNA. (C) Nanog levels remained elevated for up to 72 h after knockdown of BRG1. (D) siRNA-mediated BRG1 showed 85% knockdown efficiency. (E) Western blot analysis was carried out at 48 h posttransfection using cell lysates prepared from mouse ES cells. BRG1 protein levels were detected by using polyclonal anti-BRG1 (N15) antibody; β-TUBULIN was used as a loading control. (F) Levels of Oct4 and Nanog were increased and Sox2 levels were decreased in ES cells at 48 h posttransfection.

    Journal: BioResearch Open Access

    Article Title: BRG1 Is Required to Maintain Pluripotency of Murine Embryonic Stem Cells

    doi: 10.1089/biores.2013.0047

    Figure Lengend Snippet: Time-course analysis was performed by using shRNA directed against Brg1 . (A) Transfection of shRNA against Brg1 showed up to 90% downregulation of Brg1 in ESD3 cells compared with embryonic stem (ES) cells transfected with control shRNA. (B) Transfection of sh Brg1 led to an increase in Oct4 levels at 24 h posttransfection but a decrease in Oct4 levels at 48 and 72 h posttransfection with sh Brg1 , compared with cells transfected with control shRNA. (C) Nanog levels remained elevated for up to 72 h after knockdown of BRG1. (D) siRNA-mediated BRG1 showed 85% knockdown efficiency. (E) Western blot analysis was carried out at 48 h posttransfection using cell lysates prepared from mouse ES cells. BRG1 protein levels were detected by using polyclonal anti-BRG1 (N15) antibody; β-TUBULIN was used as a loading control. (F) Levels of Oct4 and Nanog were increased and Sox2 levels were decreased in ES cells at 48 h posttransfection.

    Article Snippet: Quantitative reverse-transcription polymerase chain reaction analysis TaqMan Assays-on-Demand was used for the detection of the following genes (TaqMan primer ID): Brg1 (Mm01151948_m1), Oct4 (Mm00658129_gH), Sox2 (Mm00488369_s1), Nanog (Mm02019550_s1), Oct11 (Mm00478284_m1), Fgf4 (Mm00438917_m1), Fgf5 (Mm00438919_m1), Gata4 (Mm00484689_m1), Meox1 (Mm00440285_m1), and T (Mm00436877_m1).

    Techniques: shRNA, Transfection, Western Blot

    BRG1 knockdown leads to the differentiation of ES cells. (A) Quantification of markers for ectoderm ( Fgf5 ), mesoderm ( Meox and T ), and endoderm ( Gata4 ) was carried out in cells transfected with control or Brg1 siRNA and grown under normal feeder-free ES cell culture conditions. Cells transfected with Brg1 siRNA showed upregulation of Fgf5 as well as downregulation of Meox1 and Gata4 expression. (B) Mouse ES cells were transfected with control or Brg1 siRNA and cultured in the presence of 1000 U/mL of leukemia inhibitory factor (LIF). ES cells transfected with control siRNA maintained the morphology characteristics of ES cells, whereas cells transfected with Brg1 siRNA showed flattened morphology as well as reduced AP staining, compared with control cells at 48 h posttransfection.

    Journal: BioResearch Open Access

    Article Title: BRG1 Is Required to Maintain Pluripotency of Murine Embryonic Stem Cells

    doi: 10.1089/biores.2013.0047

    Figure Lengend Snippet: BRG1 knockdown leads to the differentiation of ES cells. (A) Quantification of markers for ectoderm ( Fgf5 ), mesoderm ( Meox and T ), and endoderm ( Gata4 ) was carried out in cells transfected with control or Brg1 siRNA and grown under normal feeder-free ES cell culture conditions. Cells transfected with Brg1 siRNA showed upregulation of Fgf5 as well as downregulation of Meox1 and Gata4 expression. (B) Mouse ES cells were transfected with control or Brg1 siRNA and cultured in the presence of 1000 U/mL of leukemia inhibitory factor (LIF). ES cells transfected with control siRNA maintained the morphology characteristics of ES cells, whereas cells transfected with Brg1 siRNA showed flattened morphology as well as reduced AP staining, compared with control cells at 48 h posttransfection.

    Article Snippet: Quantitative reverse-transcription polymerase chain reaction analysis TaqMan Assays-on-Demand was used for the detection of the following genes (TaqMan primer ID): Brg1 (Mm01151948_m1), Oct4 (Mm00658129_gH), Sox2 (Mm00488369_s1), Nanog (Mm02019550_s1), Oct11 (Mm00478284_m1), Fgf4 (Mm00438917_m1), Fgf5 (Mm00438919_m1), Gata4 (Mm00484689_m1), Meox1 (Mm00440285_m1), and T (Mm00436877_m1).

    Techniques: Transfection, Cell Culture, Expressing, Staining

    Interaction of BRG1 with OCT4, and the role of BRG1 in cell cycle and apoptosis. (A) Co-immunoprecipitation was carried out using anti-BRG1 antibody (N-15). Goat IgG was used as an isotype-specific control. OCT4 was found to be immunoprecipitated with BRG1, revealing an interaction between the BAF complex and OCT4. Immunoprecipitation also showed that β-CATENIN interacts with the BRG1-containing complex. (B) Cell cycle analysis was performed at 48 h posttransfection after BRG1 knockdown by BrdU staining. ESD3 cells showed reduced cell number in the S phase of the cell cycle upon BRG1 knockdown compared with cells transfected with control shRNA. (C) Annexin V staining was performed to detect apoptotic cells at 72 h posttransfection. ShRNA-mediated knockdown of BRG1 in ESD3 cells showed increased apoptosis compared with cells transfected with control shRNA.

    Journal: BioResearch Open Access

    Article Title: BRG1 Is Required to Maintain Pluripotency of Murine Embryonic Stem Cells

    doi: 10.1089/biores.2013.0047

    Figure Lengend Snippet: Interaction of BRG1 with OCT4, and the role of BRG1 in cell cycle and apoptosis. (A) Co-immunoprecipitation was carried out using anti-BRG1 antibody (N-15). Goat IgG was used as an isotype-specific control. OCT4 was found to be immunoprecipitated with BRG1, revealing an interaction between the BAF complex and OCT4. Immunoprecipitation also showed that β-CATENIN interacts with the BRG1-containing complex. (B) Cell cycle analysis was performed at 48 h posttransfection after BRG1 knockdown by BrdU staining. ESD3 cells showed reduced cell number in the S phase of the cell cycle upon BRG1 knockdown compared with cells transfected with control shRNA. (C) Annexin V staining was performed to detect apoptotic cells at 72 h posttransfection. ShRNA-mediated knockdown of BRG1 in ESD3 cells showed increased apoptosis compared with cells transfected with control shRNA.

    Article Snippet: Quantitative reverse-transcription polymerase chain reaction analysis TaqMan Assays-on-Demand was used for the detection of the following genes (TaqMan primer ID): Brg1 (Mm01151948_m1), Oct4 (Mm00658129_gH), Sox2 (Mm00488369_s1), Nanog (Mm02019550_s1), Oct11 (Mm00478284_m1), Fgf4 (Mm00438917_m1), Fgf5 (Mm00438919_m1), Gata4 (Mm00484689_m1), Meox1 (Mm00440285_m1), and T (Mm00436877_m1).

    Techniques: Immunoprecipitation, Cell Cycle Assay, BrdU Staining, Transfection, shRNA, Staining

    ( A ) OncoScan Nexus Copy Number 7.5 (Nexus) view showing a diploid 46,XX array with focal abnormalities in Chromosomes 17p and 19p (arrows). ( B ) Integrative Genomics Viewer (IGV) 2.3 view of TP53 c.594_611del18 in-frame deletion of 18 nt at 25.9% variant allele fraction (VAF) ( left ). Nexus view showing mosaic copy-neutral loss of heterozygosity (CN-LOH) of Chromosome 17p13.3-p11.2 including the TP53 locus ( right ; log 2 ratio and B-allele frequency view). ( C ) IGV 2.3 view of orthogonal targeted next-generation sequencing confirmation of SMARCA4 c.1141C > T single-nucleotide variant at 88% VAF ( left ). Nexus view showing CN-LOH of Chromosome 19p13.3-19p13.2 including the SMARCA4 locus ( right ; log 2 ratio and allele peak view).

    Journal: Cold Spring Harbor Molecular Case Studies

    Article Title: Multimodal molecular analysis of an atypical small cell carcinoma of the ovary, hypercalcemic type

    doi: 10.1101/mcs.a002956

    Figure Lengend Snippet: ( A ) OncoScan Nexus Copy Number 7.5 (Nexus) view showing a diploid 46,XX array with focal abnormalities in Chromosomes 17p and 19p (arrows). ( B ) Integrative Genomics Viewer (IGV) 2.3 view of TP53 c.594_611del18 in-frame deletion of 18 nt at 25.9% variant allele fraction (VAF) ( left ). Nexus view showing mosaic copy-neutral loss of heterozygosity (CN-LOH) of Chromosome 17p13.3-p11.2 including the TP53 locus ( right ; log 2 ratio and B-allele frequency view). ( C ) IGV 2.3 view of orthogonal targeted next-generation sequencing confirmation of SMARCA4 c.1141C > T single-nucleotide variant at 88% VAF ( left ). Nexus view showing CN-LOH of Chromosome 19p13.3-19p13.2 including the SMARCA4 locus ( right ; log 2 ratio and allele peak view).

    Article Snippet: Additional test information for the “Ovarian Cancer and Rhabdoid Tumor Predisposition Syndrome via the SMARCA4 Gene” is available at www.preventiongenetics.com .

    Techniques: Variant Assay, Next-Generation Sequencing

    ( A ) Compartment profiles (the first principal components) of shSCRAM and sh SMARCA4 data for Chr 2. The A-type (open) compartments are shown in black, and the B-type (closed) compartments are shown in gray. The same color scheme was used for the gene density

    Journal: Genome Research

    Article Title: SMARCA4 regulates gene expression and higher-order chromatin structure in proliferating mammary epithelial cells

    doi: 10.1101/gr.201624.115

    Figure Lengend Snippet: ( A ) Compartment profiles (the first principal components) of shSCRAM and sh SMARCA4 data for Chr 2. The A-type (open) compartments are shown in black, and the B-type (closed) compartments are shown in gray. The same color scheme was used for the gene density

    Article Snippet: The up-regulation of genes could be an indirect effect of SMARCA4 knockdown, because in many cases, these genes were not directly bound by SMARCA4 ( E).

    Techniques:

    ( A ) Example of a ChIP-seq genome browser view of SMARCA4 binding and the input control, the shSCRAM and sh SMARCA4 RNA-seq on Chr 5, and a zoom-in on the VCAN ( Versican ) gene, which is regulated by SMARCA4, in the lower panel. The y -axis represents the

    Journal: Genome Research

    Article Title: SMARCA4 regulates gene expression and higher-order chromatin structure in proliferating mammary epithelial cells

    doi: 10.1101/gr.201624.115

    Figure Lengend Snippet: ( A ) Example of a ChIP-seq genome browser view of SMARCA4 binding and the input control, the shSCRAM and sh SMARCA4 RNA-seq on Chr 5, and a zoom-in on the VCAN ( Versican ) gene, which is regulated by SMARCA4, in the lower panel. The y -axis represents the

    Article Snippet: The up-regulation of genes could be an indirect effect of SMARCA4 knockdown, because in many cases, these genes were not directly bound by SMARCA4 ( E).

    Techniques: Chromatin Immunoprecipitation, Binding Assay, RNA Sequencing Assay

    Genome-wide all-by-all Hi-C interaction heatmaps at 1-Mb resolution and a zoom-in of Chr 11 at 250-kb resolution ( middle ) and at 40-kb resolution ( lower ) in MCF-10A shSCRAM ( A ) and MCF-10A sh SMARCA4 cells ( B ). For the genome-wide heatmaps, the chromosomes

    Journal: Genome Research

    Article Title: SMARCA4 regulates gene expression and higher-order chromatin structure in proliferating mammary epithelial cells

    doi: 10.1101/gr.201624.115

    Figure Lengend Snippet: Genome-wide all-by-all Hi-C interaction heatmaps at 1-Mb resolution and a zoom-in of Chr 11 at 250-kb resolution ( middle ) and at 40-kb resolution ( lower ) in MCF-10A shSCRAM ( A ) and MCF-10A sh SMARCA4 cells ( B ). For the genome-wide heatmaps, the chromosomes

    Article Snippet: The up-regulation of genes could be an indirect effect of SMARCA4 knockdown, because in many cases, these genes were not directly bound by SMARCA4 ( E).

    Techniques: Genome Wide, Hi-C

    ( A ) An example of a region on Chr 9 (Chr 9: 103800001–123920000) showing (from top to bottom ) the compartment profiles of sh SMARCA4 and shSCRAM at 250-kb intervals, the insulation plot profiles at 40-kb intervals (see Methods), the insulation

    Journal: Genome Research

    Article Title: SMARCA4 regulates gene expression and higher-order chromatin structure in proliferating mammary epithelial cells

    doi: 10.1101/gr.201624.115

    Figure Lengend Snippet: ( A ) An example of a region on Chr 9 (Chr 9: 103800001–123920000) showing (from top to bottom ) the compartment profiles of sh SMARCA4 and shSCRAM at 250-kb intervals, the insulation plot profiles at 40-kb intervals (see Methods), the insulation

    Article Snippet: The up-regulation of genes could be an indirect effect of SMARCA4 knockdown, because in many cases, these genes were not directly bound by SMARCA4 ( E).

    Techniques:

    ( A ) Western blot of the SMARCA4 protein levels of shSCRAM and sh SMARCA4 MCF-10A cells in the noninduced (DOX−) and induced (DOX+) conditions. Lower : Quantification of the Western blot showing ∼85% reduction of SMARCA4 protein levels upon

    Journal: Genome Research

    Article Title: SMARCA4 regulates gene expression and higher-order chromatin structure in proliferating mammary epithelial cells

    doi: 10.1101/gr.201624.115

    Figure Lengend Snippet: ( A ) Western blot of the SMARCA4 protein levels of shSCRAM and sh SMARCA4 MCF-10A cells in the noninduced (DOX−) and induced (DOX+) conditions. Lower : Quantification of the Western blot showing ∼85% reduction of SMARCA4 protein levels upon

    Article Snippet: The up-regulation of genes could be an indirect effect of SMARCA4 knockdown, because in many cases, these genes were not directly bound by SMARCA4 ( E).

    Techniques: Western Blot

    ( A ) Genome wide interaction heatmap at 2.5 Mb resolution showing the differences between interactions that are gained and lost upon SMARCA4 knockdown. The chromosomes are stacked from top-left to bottom-right in order (Chr 1, Chr 2, …, Chr 22,

    Journal: Genome Research

    Article Title: SMARCA4 regulates gene expression and higher-order chromatin structure in proliferating mammary epithelial cells

    doi: 10.1101/gr.201624.115

    Figure Lengend Snippet: ( A ) Genome wide interaction heatmap at 2.5 Mb resolution showing the differences between interactions that are gained and lost upon SMARCA4 knockdown. The chromosomes are stacked from top-left to bottom-right in order (Chr 1, Chr 2, …, Chr 22,

    Article Snippet: The up-regulation of genes could be an indirect effect of SMARCA4 knockdown, because in many cases, these genes were not directly bound by SMARCA4 ( E).

    Techniques: Genome Wide

    BRG1 and NF-κB form a DNA binding complex in myelinating cells (A) P2 or P5 mouse sciatic nerve lysates were analyzed for protein binding to DNA using a consensus sequence for NF-κB in an electrophoretic mobility shift assay (EMSA). To identify the proteins in the complex bound to the NF-κB consensus sites, specific antibodies (ab) to BRG1 (2 different antibodies were used, as indicated), p65 or a control antibody were added to the binding reaction. The complex and unbound DNA was then separated by gel electrophoresis. Excess unlabeled NF- κB consensus DNA (cold) was added as a control (n=3). Note the reduction in the binding complex caused by either BRG1 antibody and the antibody to p65. Similar EMSAs were performed on nerve lysates using consensus sequences for EGR2/Krox20 and Oct6, as indicated. (B) EMSA analysis using an NF-κB probe was performed on lysates from DRG/Schwann cell co-cultures either prior to stimulation with ascorbic acid (AA), to induce myelination, or after 6 days of AA treatment, in the presence or absence of antibodies to BRG1 or control (n=3). (C) HEK 293 cells were treated with TNFα for 1 hr to stimulate NF-κB, lysed and the lysates subjected to an EMSA using an NF-κB consensus sequence. In contrast to experiments in myelinating cells, addition of antibodies to BRG1 had no effect on the protein-DNA complex. (D) P2 rat sciatic nerve lysate was analyzed by EMSA using a NF-κB probe. Supershift analysis was used to identify DNA bound proteins. Addition of antibodies to BRM or control had no effect on protein-DNA binding. Unlabeled probe (cold) was added as a control.

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: NF-?B forms a complex with the chromatin remodeler BRG1 to regulate Schwann cell differentiation

    doi: 10.1523/JNEUROSCI.3223-12.2013

    Figure Lengend Snippet: BRG1 and NF-κB form a DNA binding complex in myelinating cells (A) P2 or P5 mouse sciatic nerve lysates were analyzed for protein binding to DNA using a consensus sequence for NF-κB in an electrophoretic mobility shift assay (EMSA). To identify the proteins in the complex bound to the NF-κB consensus sites, specific antibodies (ab) to BRG1 (2 different antibodies were used, as indicated), p65 or a control antibody were added to the binding reaction. The complex and unbound DNA was then separated by gel electrophoresis. Excess unlabeled NF- κB consensus DNA (cold) was added as a control (n=3). Note the reduction in the binding complex caused by either BRG1 antibody and the antibody to p65. Similar EMSAs were performed on nerve lysates using consensus sequences for EGR2/Krox20 and Oct6, as indicated. (B) EMSA analysis using an NF-κB probe was performed on lysates from DRG/Schwann cell co-cultures either prior to stimulation with ascorbic acid (AA), to induce myelination, or after 6 days of AA treatment, in the presence or absence of antibodies to BRG1 or control (n=3). (C) HEK 293 cells were treated with TNFα for 1 hr to stimulate NF-κB, lysed and the lysates subjected to an EMSA using an NF-κB consensus sequence. In contrast to experiments in myelinating cells, addition of antibodies to BRG1 had no effect on the protein-DNA complex. (D) P2 rat sciatic nerve lysate was analyzed by EMSA using a NF-κB probe. Supershift analysis was used to identify DNA bound proteins. Addition of antibodies to BRM or control had no effect on protein-DNA binding. Unlabeled probe (cold) was added as a control.

    Article Snippet: Ironically, these transcription factors not only target the BRG1 complex to DNA sequences, but also are dependent on BRG1 for DNA binding, since in the absence of BRG1, MyoD and MEF2 were unable to bind to the myogenin promoter ( ).

    Techniques: Binding Assay, Protein Binding, Sequencing, Electrophoretic Mobility Shift Assay, Nucleic Acid Electrophoresis

    NF-κB is required for BRG1 activation in myelinating cells (A) DRG/ Schwann cell co-cultures were stimulated for 6 days with ascorbic acid to induce myelination. Lysates were collected every two days. The NF-κB inhibitor SN50 (50μg/ml) was added to some cultures for 48 hr prior to cell lysis at the 6 day time point. Cell lysates were Western blotted for Myelin basic protein (MBP) and Tubulin. (B) Lysates from myelinating DRG/ Schwann cell co-were collected every two days. In some cultures, SN50 (50 μg/ml) was added for 48 hr prior to cell lysis at 6 days. The cell lysates were immunoprecipitated with BRG1 antibodies, subjected to ATPase assays and the amount of released Pi was quantified and normalized to the activity at day 0, prior to stimulating myelination by ascorbic acid addition. A one-way ANOVA used to calculate significance, followed by Tukey’s post hoc test. (The mean+/− SEM is shown, n=3, *p

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: NF-?B forms a complex with the chromatin remodeler BRG1 to regulate Schwann cell differentiation

    doi: 10.1523/JNEUROSCI.3223-12.2013

    Figure Lengend Snippet: NF-κB is required for BRG1 activation in myelinating cells (A) DRG/ Schwann cell co-cultures were stimulated for 6 days with ascorbic acid to induce myelination. Lysates were collected every two days. The NF-κB inhibitor SN50 (50μg/ml) was added to some cultures for 48 hr prior to cell lysis at the 6 day time point. Cell lysates were Western blotted for Myelin basic protein (MBP) and Tubulin. (B) Lysates from myelinating DRG/ Schwann cell co-were collected every two days. In some cultures, SN50 (50 μg/ml) was added for 48 hr prior to cell lysis at 6 days. The cell lysates were immunoprecipitated with BRG1 antibodies, subjected to ATPase assays and the amount of released Pi was quantified and normalized to the activity at day 0, prior to stimulating myelination by ascorbic acid addition. A one-way ANOVA used to calculate significance, followed by Tukey’s post hoc test. (The mean+/− SEM is shown, n=3, *p

    Article Snippet: Ironically, these transcription factors not only target the BRG1 complex to DNA sequences, but also are dependent on BRG1 for DNA binding, since in the absence of BRG1, MyoD and MEF2 were unable to bind to the myogenin promoter ( ).

    Techniques: Activation Assay, Lysis, Western Blot, Immunoprecipitation, Activity Assay

    Neuregulin I (NRG1) type III stimulates the association of BRG1 with NF-κB and elevates BRG1 ATPase activity (A) Isolated rat Schwann cells were left untreated or treated with either untransfected cell membranes (UnT) or with cell membranes isolated from HEK 293 cells transfected with NRG1 type III (NRG). Following 1 hour of membrane treatment, the Schwann cells were rinsed, lysed and subjected to immunoprecipitiation with BRG1 or p65 antibodies or with an IgG control (Ig). Immunoprecipitates were analyzed by Western blotting using anti-BRG1 or anti-p65. Quantification of the Western blots is shown to the right. A two-tailed Student’s t-test used to calculate significance. The mean+/− SEM is shown, n=3. *p

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: NF-?B forms a complex with the chromatin remodeler BRG1 to regulate Schwann cell differentiation

    doi: 10.1523/JNEUROSCI.3223-12.2013

    Figure Lengend Snippet: Neuregulin I (NRG1) type III stimulates the association of BRG1 with NF-κB and elevates BRG1 ATPase activity (A) Isolated rat Schwann cells were left untreated or treated with either untransfected cell membranes (UnT) or with cell membranes isolated from HEK 293 cells transfected with NRG1 type III (NRG). Following 1 hour of membrane treatment, the Schwann cells were rinsed, lysed and subjected to immunoprecipitiation with BRG1 or p65 antibodies or with an IgG control (Ig). Immunoprecipitates were analyzed by Western blotting using anti-BRG1 or anti-p65. Quantification of the Western blots is shown to the right. A two-tailed Student’s t-test used to calculate significance. The mean+/− SEM is shown, n=3. *p

    Article Snippet: Ironically, these transcription factors not only target the BRG1 complex to DNA sequences, but also are dependent on BRG1 for DNA binding, since in the absence of BRG1, MyoD and MEF2 were unable to bind to the myogenin promoter ( ).

    Techniques: Activity Assay, Isolation, Transfection, Western Blot, Two Tailed Test

    BRG1 is required for Schwann cell differentiation in response to elevated cAMP (A) Primary rat Schwann cells were transfected with siRNA to BRG1 or control siRNA overnight and then stimulated with 500μM dbCAMP for 72 hours. Cells were lysed and analyzed by Western blotting for BRG1, OCT6, and α-tubulin. [The image shown is from the same blot, but the order of the lanes was altered for the figure, as indicated by the line.] (B) Quantitative analysis of (A) was performed using Scion image quant and the results were normalized to the untransfected signal in each experiment (UnT). A one-way ANOVA was used to calculate significance, followed by a Tukey’s post hoc test (n=4).

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: NF-?B forms a complex with the chromatin remodeler BRG1 to regulate Schwann cell differentiation

    doi: 10.1523/JNEUROSCI.3223-12.2013

    Figure Lengend Snippet: BRG1 is required for Schwann cell differentiation in response to elevated cAMP (A) Primary rat Schwann cells were transfected with siRNA to BRG1 or control siRNA overnight and then stimulated with 500μM dbCAMP for 72 hours. Cells were lysed and analyzed by Western blotting for BRG1, OCT6, and α-tubulin. [The image shown is from the same blot, but the order of the lanes was altered for the figure, as indicated by the line.] (B) Quantitative analysis of (A) was performed using Scion image quant and the results were normalized to the untransfected signal in each experiment (UnT). A one-way ANOVA was used to calculate significance, followed by a Tukey’s post hoc test (n=4).

    Article Snippet: Ironically, these transcription factors not only target the BRG1 complex to DNA sequences, but also are dependent on BRG1 for DNA binding, since in the absence of BRG1, MyoD and MEF2 were unable to bind to the myogenin promoter ( ).

    Techniques: Cell Differentiation, Transfection, Western Blot

    BRG1 cko mice display severe hypomyelination (A) A Kaplan-Meier survival curve indicating the survival of wild type (CTR) or Brg1 F/F ;Dhh-Cre (Brg1CKO) mice (n=31). (B) Image of wild type (CTR) and BRG conditional knockout (Brg1CKO) sciatic nerves at p11. (C) Electron micrographs of cross sections of Control, Brg1 F/+ ;Dhh-Cre (CTR) and Brg1 F/F ;Dhh-Cre (Brg1CKO) nerves at P7 (upper panels) and P18 (lower panels). The asterisks indicate atypical bundles of axons and the arrow points out a a single Schwann cell surrounding an unmyelinated axon. Note the complete lack of myelin profiles in the cko nerves.

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: NF-?B forms a complex with the chromatin remodeler BRG1 to regulate Schwann cell differentiation

    doi: 10.1523/JNEUROSCI.3223-12.2013

    Figure Lengend Snippet: BRG1 cko mice display severe hypomyelination (A) A Kaplan-Meier survival curve indicating the survival of wild type (CTR) or Brg1 F/F ;Dhh-Cre (Brg1CKO) mice (n=31). (B) Image of wild type (CTR) and BRG conditional knockout (Brg1CKO) sciatic nerves at p11. (C) Electron micrographs of cross sections of Control, Brg1 F/+ ;Dhh-Cre (CTR) and Brg1 F/F ;Dhh-Cre (Brg1CKO) nerves at P7 (upper panels) and P18 (lower panels). The asterisks indicate atypical bundles of axons and the arrow points out a a single Schwann cell surrounding an unmyelinated axon. Note the complete lack of myelin profiles in the cko nerves.

    Article Snippet: Ironically, these transcription factors not only target the BRG1 complex to DNA sequences, but also are dependent on BRG1 for DNA binding, since in the absence of BRG1, MyoD and MEF2 were unable to bind to the myogenin promoter ( ).

    Techniques: Mouse Assay, Knock-Out

    Conditional deletion of BRG1 results in the loss in the expression of promyelinating transcription factors (A) Control, Brg1 F/+ ;Dhh-Cre , (CTR) and Brg1 F/F ;Dhh-Cre (BRG1CKO) sciatic nerves from P7 were fixed, cryosectioned and immunostained for EGR2/Krox20, Oct6, or Sox2 (red) or Sox10 (red) and Ki67 (green). The nuclei were labeled with Topro3 (blue). (B) Cryosections were immunostained with antibodies to the cleaved form of caspase 3 (green) and Sox10 (red) and the nuclei were labeled with Topro3 (blue). The boxed area is depicted enlarged on the right (B′) where a couple of Sox10+ cells with activated caspase 3 are indicated by the arrows. (C) The fraction of Topro3 labeled cells that were positive for each of the indicated markers was quantified and a student’s t-test was used to calculate significance. (Mean+/− SEM is shown, n=3. *p

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: NF-?B forms a complex with the chromatin remodeler BRG1 to regulate Schwann cell differentiation

    doi: 10.1523/JNEUROSCI.3223-12.2013

    Figure Lengend Snippet: Conditional deletion of BRG1 results in the loss in the expression of promyelinating transcription factors (A) Control, Brg1 F/+ ;Dhh-Cre , (CTR) and Brg1 F/F ;Dhh-Cre (BRG1CKO) sciatic nerves from P7 were fixed, cryosectioned and immunostained for EGR2/Krox20, Oct6, or Sox2 (red) or Sox10 (red) and Ki67 (green). The nuclei were labeled with Topro3 (blue). (B) Cryosections were immunostained with antibodies to the cleaved form of caspase 3 (green) and Sox10 (red) and the nuclei were labeled with Topro3 (blue). The boxed area is depicted enlarged on the right (B′) where a couple of Sox10+ cells with activated caspase 3 are indicated by the arrows. (C) The fraction of Topro3 labeled cells that were positive for each of the indicated markers was quantified and a student’s t-test was used to calculate significance. (Mean+/− SEM is shown, n=3. *p

    Article Snippet: Ironically, these transcription factors not only target the BRG1 complex to DNA sequences, but also are dependent on BRG1 for DNA binding, since in the absence of BRG1, MyoD and MEF2 were unable to bind to the myogenin promoter ( ).

    Techniques: Expressing, Labeling

    BRG1 and NF-κB complex at NF-κB-DNA binding sites in myelinating cells ), (n=3). The NF-κB binding site at −1 to −176 of the Sox10 gene is the solid box in the diagram. A sample PCR reaction is shown and the quantification relative to the control, IgG antibody, is shown on the right (n=3). A one-way ANOVA was used to calculate significance, followed by Tukey’s post hoc test. (The mean+/−SEM is shown. *p

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: NF-?B forms a complex with the chromatin remodeler BRG1 to regulate Schwann cell differentiation

    doi: 10.1523/JNEUROSCI.3223-12.2013

    Figure Lengend Snippet: BRG1 and NF-κB complex at NF-κB-DNA binding sites in myelinating cells ), (n=3). The NF-κB binding site at −1 to −176 of the Sox10 gene is the solid box in the diagram. A sample PCR reaction is shown and the quantification relative to the control, IgG antibody, is shown on the right (n=3). A one-way ANOVA was used to calculate significance, followed by Tukey’s post hoc test. (The mean+/−SEM is shown. *p

    Article Snippet: Ironically, these transcription factors not only target the BRG1 complex to DNA sequences, but also are dependent on BRG1 for DNA binding, since in the absence of BRG1, MyoD and MEF2 were unable to bind to the myogenin promoter ( ).

    Techniques: Binding Assay, Polymerase Chain Reaction

    BRG1 is expressed and activated in myelinating rat sciatic nerves (A) Sciatic nerves from rats at postnatal day (p) 1, 3, 5, 7, and adult (approximately 3–4 months of age) were analyzed by Western blot for BRG1 and α-tubulin, as a loading control. The level of expression was quantified and the graph indicates BRG1 relative units as compared to postnatal day 1. To determine statistical significance a one way ANOVA was performed followed by Tukey’s post hoc test. (B) P5 sciatic nerves were fixed, cryosectioned and immunostained with antibodies to BRG1 and Sox10. (C) BRG1 ATPase activity was measured in P3, 5, and 7 rat sciatic nerve lysates by immunoprecipitation of BRG1 (right panel) followed by an in vitro ATPase assay using radiolabelled ATP. The released Pi was separated from ATP using TLC. Positive(+) and negative(−) controls were performed with lysate from E15 rat embryos (Bultman, 2005) with or without DNA added to the reaction mixture, respectively. (D) The intensity of the signal was quantified by densitometry. Statistical significance was performed by a one way ANOVA followed by a Tukey’s post hoc test (n=3).

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: NF-?B forms a complex with the chromatin remodeler BRG1 to regulate Schwann cell differentiation

    doi: 10.1523/JNEUROSCI.3223-12.2013

    Figure Lengend Snippet: BRG1 is expressed and activated in myelinating rat sciatic nerves (A) Sciatic nerves from rats at postnatal day (p) 1, 3, 5, 7, and adult (approximately 3–4 months of age) were analyzed by Western blot for BRG1 and α-tubulin, as a loading control. The level of expression was quantified and the graph indicates BRG1 relative units as compared to postnatal day 1. To determine statistical significance a one way ANOVA was performed followed by Tukey’s post hoc test. (B) P5 sciatic nerves were fixed, cryosectioned and immunostained with antibodies to BRG1 and Sox10. (C) BRG1 ATPase activity was measured in P3, 5, and 7 rat sciatic nerve lysates by immunoprecipitation of BRG1 (right panel) followed by an in vitro ATPase assay using radiolabelled ATP. The released Pi was separated from ATP using TLC. Positive(+) and negative(−) controls were performed with lysate from E15 rat embryos (Bultman, 2005) with or without DNA added to the reaction mixture, respectively. (D) The intensity of the signal was quantified by densitometry. Statistical significance was performed by a one way ANOVA followed by a Tukey’s post hoc test (n=3).

    Article Snippet: Ironically, these transcription factors not only target the BRG1 complex to DNA sequences, but also are dependent on BRG1 for DNA binding, since in the absence of BRG1, MyoD and MEF2 were unable to bind to the myogenin promoter ( ).

    Techniques: Western Blot, Expressing, Activity Assay, Immunoprecipitation, In Vitro, ATPase Assay, Thin Layer Chromatography

    BRG1 controls dynamics of MITF binding. ( A ) ChIP-qPCR of 3HA-MITF in 501Mel-CL8 cells at the indicated loci following transfection with siLuc or siBRG1. The protamine 1 locus (PRM1) was used as a negative control. ( B ) UCSC screenshots illustrating binding of MITF between two BRG1-occupied nucleosomes at selected loci assayed by ChIP-qPCR in panel A . sThe GPR110-1 and GPR110-2 sites assayed in Panel A are indicated in panel B . ( C ) A model for regulatory elements in the melanocyte lineage. Melanocyte lineage enhancers comprise combinations of MITF, SOX10, YY1, and also TFAP2A and ETS1 (not represented for simplicity. Note also that Pol II and the PIC are present at active enhancers where enhancer RNAs are made. For simplicity these are also not represented.) bound to a nucleosome-depleted region. MITF but also these other factors recruit BRG1/PBAF to the nucleosomes flanking the combinations of transcription factors. BRG1/PBAF also occupies the nucleosomes flanking the TSS and a subset of these promoters further comprises a MITF binding site close to the TSS. DOI: http://dx.doi.org/10.7554/eLife.06857.018

    Journal: eLife

    Article Title: Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells

    doi: 10.7554/eLife.06857

    Figure Lengend Snippet: BRG1 controls dynamics of MITF binding. ( A ) ChIP-qPCR of 3HA-MITF in 501Mel-CL8 cells at the indicated loci following transfection with siLuc or siBRG1. The protamine 1 locus (PRM1) was used as a negative control. ( B ) UCSC screenshots illustrating binding of MITF between two BRG1-occupied nucleosomes at selected loci assayed by ChIP-qPCR in panel A . sThe GPR110-1 and GPR110-2 sites assayed in Panel A are indicated in panel B . ( C ) A model for regulatory elements in the melanocyte lineage. Melanocyte lineage enhancers comprise combinations of MITF, SOX10, YY1, and also TFAP2A and ETS1 (not represented for simplicity. Note also that Pol II and the PIC are present at active enhancers where enhancer RNAs are made. For simplicity these are also not represented.) bound to a nucleosome-depleted region. MITF but also these other factors recruit BRG1/PBAF to the nucleosomes flanking the combinations of transcription factors. BRG1/PBAF also occupies the nucleosomes flanking the TSS and a subset of these promoters further comprises a MITF binding site close to the TSS. DOI: http://dx.doi.org/10.7554/eLife.06857.018

    Article Snippet: Excel spread sheet of genes specifically and commonly regulated by BRG1 and MITF knockdown in 501Mel and Hermes 3A cells along with the appropriate gene ontology, see Figures S3B–D.

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

    Composition of BRG1 complexes in 501Mel cells. ( A ) BRG1 associates with CHD7 in 501Mel cells. Following immunoprecipitation of 501Mel cell extracts with anti-BRG1 antibody or HA beads as control the eluted fractions were probed with antibodies for the indicated proteins. ( B ) Following immunoprecipitation of 501Mel cell extracts with anti-CHD7 antibody or HA beads as control the eluted fractions were probed with antibodies for the indicated proteins. ( C ) Table summarising the known subunits of BRG1-containing complexes highlighting catalytic subunits with ATPase activity, common subunits and specific subunits. The composition of the complex interacting with MITF based on mass-spectrometry and immunoblots is schematised. DOI: http://dx.doi.org/10.7554/eLife.06857.004

    Journal: eLife

    Article Title: Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells

    doi: 10.7554/eLife.06857

    Figure Lengend Snippet: Composition of BRG1 complexes in 501Mel cells. ( A ) BRG1 associates with CHD7 in 501Mel cells. Following immunoprecipitation of 501Mel cell extracts with anti-BRG1 antibody or HA beads as control the eluted fractions were probed with antibodies for the indicated proteins. ( B ) Following immunoprecipitation of 501Mel cell extracts with anti-CHD7 antibody or HA beads as control the eluted fractions were probed with antibodies for the indicated proteins. ( C ) Table summarising the known subunits of BRG1-containing complexes highlighting catalytic subunits with ATPase activity, common subunits and specific subunits. The composition of the complex interacting with MITF based on mass-spectrometry and immunoblots is schematised. DOI: http://dx.doi.org/10.7554/eLife.06857.004

    Article Snippet: Excel spread sheet of genes specifically and commonly regulated by BRG1 and MITF knockdown in 501Mel and Hermes 3A cells along with the appropriate gene ontology, see Figures S3B–D.

    Techniques: Immunoprecipitation, Activity Assay, Mass Spectrometry, Western Blot

    BRG1 and MITF repress gene expression. ( A – B ) UCSC screenshots illustrating that MITF recruits BRG1 to regulatory elements of the SERPINE1 and IL24 loci. BRG1 is diminished at these genes in siMITF-silenced cells where these genes are activated. ( C ) BRG1 re-localizes over the genome in siMITF senescent cells. UCSC screenshot illustrating that BRG1 occupancy is strongly increased over the CCL2 locus in siMITF-silenced cells where this gene is strongly activated. DOI: http://dx.doi.org/10.7554/eLife.06857.017

    Journal: eLife

    Article Title: Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells

    doi: 10.7554/eLife.06857

    Figure Lengend Snippet: BRG1 and MITF repress gene expression. ( A – B ) UCSC screenshots illustrating that MITF recruits BRG1 to regulatory elements of the SERPINE1 and IL24 loci. BRG1 is diminished at these genes in siMITF-silenced cells where these genes are activated. ( C ) BRG1 re-localizes over the genome in siMITF senescent cells. UCSC screenshot illustrating that BRG1 occupancy is strongly increased over the CCL2 locus in siMITF-silenced cells where this gene is strongly activated. DOI: http://dx.doi.org/10.7554/eLife.06857.017

    Article Snippet: Excel spread sheet of genes specifically and commonly regulated by BRG1 and MITF knockdown in 501Mel and Hermes 3A cells along with the appropriate gene ontology, see Figures S3B–D.

    Techniques: Expressing

    Profiling of BRG1 genome occupancy. ( A – B ) UCSC screenshots illustrating BRG1 occupancy over the ARRDC2 and REL1 loci highlighting co-localization with H3K27ac-marked enhancers either in the Encode data track or in Human Foreskin Melanocytes (HFM). ( C ) Enrichment of TF binding motifs at BRG1-occupied sites. ( D ) Clustering analysis of BRG1 occupancy at RefSeq TSS illustrating the presence of BRG1 at a subset of TSS with different binding profiles. Cluster E showing BRG1 occupancy both upstream and downstream of the TSS was re-clustered to highlight the different profiles as shown in Figure 4E . ( E ) Integrative analysis of BRG1 ChIP-seq data with shBRG1 RNA-seq data. A table shows the number of genes with BRG1-occupied sites ±10 kb or ± 30 kb from the TSS. Venn diagrams indicate the number of these genes that are up- or down-regulated following shBRG1 knockdown. ( F ) Ontology analysis of the BRG1-associated up- and down-regulated genes. DOI: http://dx.doi.org/10.7554/eLife.06857.010

    Journal: eLife

    Article Title: Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells

    doi: 10.7554/eLife.06857

    Figure Lengend Snippet: Profiling of BRG1 genome occupancy. ( A – B ) UCSC screenshots illustrating BRG1 occupancy over the ARRDC2 and REL1 loci highlighting co-localization with H3K27ac-marked enhancers either in the Encode data track or in Human Foreskin Melanocytes (HFM). ( C ) Enrichment of TF binding motifs at BRG1-occupied sites. ( D ) Clustering analysis of BRG1 occupancy at RefSeq TSS illustrating the presence of BRG1 at a subset of TSS with different binding profiles. Cluster E showing BRG1 occupancy both upstream and downstream of the TSS was re-clustered to highlight the different profiles as shown in Figure 4E . ( E ) Integrative analysis of BRG1 ChIP-seq data with shBRG1 RNA-seq data. A table shows the number of genes with BRG1-occupied sites ±10 kb or ± 30 kb from the TSS. Venn diagrams indicate the number of these genes that are up- or down-regulated following shBRG1 knockdown. ( F ) Ontology analysis of the BRG1-associated up- and down-regulated genes. DOI: http://dx.doi.org/10.7554/eLife.06857.010

    Article Snippet: Excel spread sheet of genes specifically and commonly regulated by BRG1 and MITF knockdown in 501Mel and Hermes 3A cells along with the appropriate gene ontology, see Figures S3B–D.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, RNA Sequencing Assay

    TF binding motifs at MITF and BRG1-bound sites. ( A ) Ontology analysis of genes associated with BRG1-MITF-co-occupied sites. ( B ) Identification of transcription factor binding motifs at BRG1-MITF-occupied sites. MEME-ChIP identified several motifs enriched at these sites. The most prominent are the E-box and M-box corresponding to MITF binding, SOX10, CREB, and ETS1 as well as motifs with no known annotation. ( C ) Identification of transcription factor binding motifs at BRG1-occupied sites that are strongly enriched in siMITF senescent cells. ( C – D ) Identification of transcription factor binding motifs at MITF-occupied sites that are associated with down and up-regulated genes after MITF silencing. DOI: http://dx.doi.org/10.7554/eLife.06857.012

    Journal: eLife

    Article Title: Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells

    doi: 10.7554/eLife.06857

    Figure Lengend Snippet: TF binding motifs at MITF and BRG1-bound sites. ( A ) Ontology analysis of genes associated with BRG1-MITF-co-occupied sites. ( B ) Identification of transcription factor binding motifs at BRG1-MITF-occupied sites. MEME-ChIP identified several motifs enriched at these sites. The most prominent are the E-box and M-box corresponding to MITF binding, SOX10, CREB, and ETS1 as well as motifs with no known annotation. ( C ) Identification of transcription factor binding motifs at BRG1-occupied sites that are strongly enriched in siMITF senescent cells. ( C – D ) Identification of transcription factor binding motifs at MITF-occupied sites that are associated with down and up-regulated genes after MITF silencing. DOI: http://dx.doi.org/10.7554/eLife.06857.012

    Article Snippet: Excel spread sheet of genes specifically and commonly regulated by BRG1 and MITF knockdown in 501Mel and Hermes 3A cells along with the appropriate gene ontology, see Figures S3B–D.

    Techniques: Binding Assay, Chromatin Immunoprecipitation

    Identification of YY1-TFAP2A associated MAREs. ( A ) Clustering at YY1 occupied sites illustrates co-localization with BRG1. ( B ) Clustering at TFAP2A occupied sites illustrates co-localization with BRG1 and H3K27ac. ( C ) Identifcation of TFAP2A sites co-occupied by MITF. The re-clustering shown to the right illustrates that MITF may be localized at various distances up- or down-stream of TFAP2A. ( D ) Clustering identifies MAREs where MITF co-localizes with YY1, TFAP2A, BRG1, and H3K27ac. DOI: http://dx.doi.org/10.7554/eLife.06857.015

    Journal: eLife

    Article Title: Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells

    doi: 10.7554/eLife.06857

    Figure Lengend Snippet: Identification of YY1-TFAP2A associated MAREs. ( A ) Clustering at YY1 occupied sites illustrates co-localization with BRG1. ( B ) Clustering at TFAP2A occupied sites illustrates co-localization with BRG1 and H3K27ac. ( C ) Identifcation of TFAP2A sites co-occupied by MITF. The re-clustering shown to the right illustrates that MITF may be localized at various distances up- or down-stream of TFAP2A. ( D ) Clustering identifies MAREs where MITF co-localizes with YY1, TFAP2A, BRG1, and H3K27ac. DOI: http://dx.doi.org/10.7554/eLife.06857.015

    Article Snippet: Excel spread sheet of genes specifically and commonly regulated by BRG1 and MITF knockdown in 501Mel and Hermes 3A cells along with the appropriate gene ontology, see Figures S3B–D.

    Techniques:

    BRG1 is essential in mouse melanocytes in vivo. ( A ) Statistics relevant to the phenotype of the mice lacking Brg1 in the melanocyte lineage. Note that as the Tyr -Cre transgene is present on the X chromosome, we detect black mice with the Tyr -Cre:: Smarca4 lox/lox genotype, but in these animals the Cre-recombinase is subjected to X-inactivation such that recombination does not occur in all melanoblasts. ( B ) Photographs of representative mice of the indicated genotypes. Bright field images of hair from the backs of 8-week-old animals of the two genotypes are also shown. Magnification X40. ( C ) Labelling of dorsal hair follicle bulbs with antibodies against Sox10 in red and Dct in green along with Hoechst-stained nuclei in blue. Arrows indicate labelled melanocytes in the bulb of Brg1 expressing mice. Scale bars are 50 μm. DOI: http://dx.doi.org/10.7554/eLife.06857.008

    Journal: eLife

    Article Title: Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells

    doi: 10.7554/eLife.06857

    Figure Lengend Snippet: BRG1 is essential in mouse melanocytes in vivo. ( A ) Statistics relevant to the phenotype of the mice lacking Brg1 in the melanocyte lineage. Note that as the Tyr -Cre transgene is present on the X chromosome, we detect black mice with the Tyr -Cre:: Smarca4 lox/lox genotype, but in these animals the Cre-recombinase is subjected to X-inactivation such that recombination does not occur in all melanoblasts. ( B ) Photographs of representative mice of the indicated genotypes. Bright field images of hair from the backs of 8-week-old animals of the two genotypes are also shown. Magnification X40. ( C ) Labelling of dorsal hair follicle bulbs with antibodies against Sox10 in red and Dct in green along with Hoechst-stained nuclei in blue. Arrows indicate labelled melanocytes in the bulb of Brg1 expressing mice. Scale bars are 50 μm. DOI: http://dx.doi.org/10.7554/eLife.06857.008

    Article Snippet: Excel spread sheet of genes specifically and commonly regulated by BRG1 and MITF knockdown in 501Mel and Hermes 3A cells along with the appropriate gene ontology, see Figures S3B–D.

    Techniques: In Vivo, Mouse Assay, Staining, Expressing

    SOX10 regulates MITF expression in 501Mel cells. ( A ) Western blot analysis of MITF expression in the indicated cell types after siRNA transfections. ( B ) RT-qPCR analysis of gene expression in the indicated cell types after siRNA transfections. ( C ) Western blot analysis of BRG1 and MITF expression in 501Mel CL8 cells following siRNA transfections. Phase contrast microscopy of 501Mel Cl8 cells following siRNA transfections. Magnification X10. ( D ) RT-qPCR analysis of gene expression in the indicated cell types after siRNA transfections. DOI: http://dx.doi.org/10.7554/eLife.06857.007

    Journal: eLife

    Article Title: Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells

    doi: 10.7554/eLife.06857

    Figure Lengend Snippet: SOX10 regulates MITF expression in 501Mel cells. ( A ) Western blot analysis of MITF expression in the indicated cell types after siRNA transfections. ( B ) RT-qPCR analysis of gene expression in the indicated cell types after siRNA transfections. ( C ) Western blot analysis of BRG1 and MITF expression in 501Mel CL8 cells following siRNA transfections. Phase contrast microscopy of 501Mel Cl8 cells following siRNA transfections. Magnification X10. ( D ) RT-qPCR analysis of gene expression in the indicated cell types after siRNA transfections. DOI: http://dx.doi.org/10.7554/eLife.06857.007

    Article Snippet: Excel spread sheet of genes specifically and commonly regulated by BRG1 and MITF knockdown in 501Mel and Hermes 3A cells along with the appropriate gene ontology, see Figures S3B–D.

    Techniques: Expressing, Western Blot, Transfection, Quantitative RT-PCR, Microscopy

    Purification of MITF-associated complexes. ( A ) Western blot of 501Mel cell lines stably expressing Flag-HA-tagged-MITF (F-H-MITF). ( B ) The immunoprecipitated material from the soluble nuclear extract (SNE) was separated by SDS PAGE and stained with silver nitrate. F-H-MITF is indicated along with * that designates a contaminating protein seen in the control immunoprecipitations. Lane M corresponds to a molecular mass marker indicated in kDa. ( C ) Immunoblot detection of HERC2, BRG1, USP7, USP11, XRCC5, and XRCC6 in the MITF-associated complexes. ( D ) Summary of proteins and complexes interacting with MITF. Shown are the proteins found specifically in the immunopurifications of F-H-MITF classified according to their function and organisation into known complexes. DOI: http://dx.doi.org/10.7554/eLife.06857.003

    Journal: eLife

    Article Title: Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells

    doi: 10.7554/eLife.06857

    Figure Lengend Snippet: Purification of MITF-associated complexes. ( A ) Western blot of 501Mel cell lines stably expressing Flag-HA-tagged-MITF (F-H-MITF). ( B ) The immunoprecipitated material from the soluble nuclear extract (SNE) was separated by SDS PAGE and stained with silver nitrate. F-H-MITF is indicated along with * that designates a contaminating protein seen in the control immunoprecipitations. Lane M corresponds to a molecular mass marker indicated in kDa. ( C ) Immunoblot detection of HERC2, BRG1, USP7, USP11, XRCC5, and XRCC6 in the MITF-associated complexes. ( D ) Summary of proteins and complexes interacting with MITF. Shown are the proteins found specifically in the immunopurifications of F-H-MITF classified according to their function and organisation into known complexes. DOI: http://dx.doi.org/10.7554/eLife.06857.003

    Article Snippet: Excel spread sheet of genes specifically and commonly regulated by BRG1 and MITF knockdown in 501Mel and Hermes 3A cells along with the appropriate gene ontology, see Figures S3B–D.

    Techniques: Purification, Western Blot, Stable Transfection, Expressing, Immunoprecipitation, SDS Page, Staining, Marker

    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: To knock down BRG1, the cells were transduced with lentiviruses expressing short hairpin RNA (shRNA) directed against BRG1 (clone 15549; Expression Arrest-RNAi Consortium human shRNA library purchased from Open Biosystems) for 4 days.

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