Structured Review

Abcam brg1 antibody
Loss of <t>BRG1</t> results in ssDNA accumulation and retention of RPA foci. (A) U2OS cells transfected with BRG1 siRNA (siBRG1) or control siRNA (siCont) were pre-labelled with BrdU and then treated with 10 µM ETO for 20 min. After 2 h,
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1) Product Images from "BRG1 promotes the repair of DNA double-strand breaks by facilitating the replacement of RPA with RAD51"

Article Title: BRG1 promotes the repair of DNA double-strand breaks by facilitating the replacement of RPA with RAD51

Journal:

doi: 10.1242/jcs.159103

Loss of BRG1 results in ssDNA accumulation and retention of RPA foci. (A) U2OS cells transfected with BRG1 siRNA (siBRG1) or control siRNA (siCont) were pre-labelled with BrdU and then treated with 10 µM ETO for 20 min. After 2 h,
Figure Legend Snippet: Loss of BRG1 results in ssDNA accumulation and retention of RPA foci. (A) U2OS cells transfected with BRG1 siRNA (siBRG1) or control siRNA (siCont) were pre-labelled with BrdU and then treated with 10 µM ETO for 20 min. After 2 h,

Techniques Used: Recombinase Polymerase Amplification, Transfection

BRG1 is a vital factor in the DDR. (A) SW13 cells transfected with pBJ5-BRG1 or empty vector and (B) U2OS cells transfected with BRG1 siRNA (siBRG1) or control siRNA (siCont) were mock treated or exposed to the indicated doses of ETO for 20 min
Figure Legend Snippet: BRG1 is a vital factor in the DDR. (A) SW13 cells transfected with pBJ5-BRG1 or empty vector and (B) U2OS cells transfected with BRG1 siRNA (siBRG1) or control siRNA (siCont) were mock treated or exposed to the indicated doses of ETO for 20 min

Techniques Used: Transfection, Plasmid Preparation

BRG1 is enriched on the chromatin at DSBs. (A) U2OS cells were incubated with 10 µM ETO and then allowed to repair DSBs. The chromatin fractions were extracted from cell pellets collected at the indicated time-points and were subjected
Figure Legend Snippet: BRG1 is enriched on the chromatin at DSBs. (A) U2OS cells were incubated with 10 µM ETO and then allowed to repair DSBs. The chromatin fractions were extracted from cell pellets collected at the indicated time-points and were subjected

Techniques Used: Incubation

A model for the role of BRG1 in regulating DSB repair. When DSBs occur, BRG1 is recruited to the DNA damage sites. Subsequently, BRG1 interacts with RAD52 and promotes its recruitment to DSB sites, which facilitates RAD51 assembly on RPA-bound ssDNAs.
Figure Legend Snippet: A model for the role of BRG1 in regulating DSB repair. When DSBs occur, BRG1 is recruited to the DNA damage sites. Subsequently, BRG1 interacts with RAD52 and promotes its recruitment to DSB sites, which facilitates RAD51 assembly on RPA-bound ssDNAs.

Techniques Used: Recombinase Polymerase Amplification

BRG1 is required for RAD51 recruitment to DNA damage sites. (A,B) U2OS cells transfected with BRG1 siRNA (siBRG1) or control siRNA (siCont) (A) and SW13 cells transfected with pBJ5-BRG1 or empty vector (B) were treated with ETO for 20 min. At
Figure Legend Snippet: BRG1 is required for RAD51 recruitment to DNA damage sites. (A,B) U2OS cells transfected with BRG1 siRNA (siBRG1) or control siRNA (siCont) (A) and SW13 cells transfected with pBJ5-BRG1 or empty vector (B) were treated with ETO for 20 min. At

Techniques Used: Transfection, Plasmid Preparation

BRG1 interacts with RAD52 and regulates its accumulation at DSB sites during homologous recombination repair. (A) U2OS cells transfected with BRCA2 siRNA (siBRCA2), RAD52 siRNA (siRAD52) or control siRNA (siCont) were exposed to 10 µM
Figure Legend Snippet: BRG1 interacts with RAD52 and regulates its accumulation at DSB sites during homologous recombination repair. (A) U2OS cells transfected with BRCA2 siRNA (siBRCA2), RAD52 siRNA (siRAD52) or control siRNA (siCont) were exposed to 10 µM

Techniques Used: Homologous Recombination, Transfection

BRG1 depletion impairs homologous recombination repair. (A) Schematic of the DR-GFP reporter used to monitor homologous recombination (HR). (B) Schematic of the EJ5-GFP reporter used to monitor NHEJ in U2OS cells (see text for details). puro, puromycin
Figure Legend Snippet: BRG1 depletion impairs homologous recombination repair. (A) Schematic of the DR-GFP reporter used to monitor homologous recombination (HR). (B) Schematic of the EJ5-GFP reporter used to monitor NHEJ in U2OS cells (see text for details). puro, puromycin

Techniques Used: Homologous Recombination, Non-Homologous End Joining

2) Product Images from "BRG1 Promotes chromatin remodeling around DNA damage sites"

Article Title: BRG1 Promotes chromatin remodeling around DNA damage sites

Journal: Animal Cells and Systems

doi: 10.1080/19768354.2018.1525429

BRG1 promotes H2AX phosphorylation. (A) SW13 cells were transfected with the pBJ5 vector or pBJ5-BRG1 plasmids for 48 h. Then, the cells were treated with 10 μM ETO for 20 min and allowed to repair for the indicated time. The whole cell lysates were detected by western blotting with the indicated antibodies. (B) U2OS cells were transfected with control siRNA or BRG1 siRNA for 48 h. Then, the cells were treated with 10 μM ETO for 20 min and allowed to repair for the indicated time. The whole cell lysates were detected by western blotting with the indicated antibodies.
Figure Legend Snippet: BRG1 promotes H2AX phosphorylation. (A) SW13 cells were transfected with the pBJ5 vector or pBJ5-BRG1 plasmids for 48 h. Then, the cells were treated with 10 μM ETO for 20 min and allowed to repair for the indicated time. The whole cell lysates were detected by western blotting with the indicated antibodies. (B) U2OS cells were transfected with control siRNA or BRG1 siRNA for 48 h. Then, the cells were treated with 10 μM ETO for 20 min and allowed to repair for the indicated time. The whole cell lysates were detected by western blotting with the indicated antibodies.

Techniques Used: Transfection, Plasmid Preparation, Western Blot

BRG1 promotes H2A release from chromatin near DSB sites. (A, B) H2A ChIP assays were performed on AsiSI-ER-U2OS cells treated with 4OHT or not for 4 h; the products were assessed by real-time q-PCR amplification using primers flanking the chr1_6:89231183 and chr6_12:90404906 DSB sites. Each value represents the mean ± S.D. of three independent experiments. (C, D) H2A ChIP assays were performed on AsiSI-ER-U2OS cells after treatment with 4OHT for 4 h or not; the products were assessed by real-time q-PCR amplification using primers near or distal to the chr22:19180307 DSB site. Each value represents the mean ± S.D. of three independent experiments.
Figure Legend Snippet: BRG1 promotes H2A release from chromatin near DSB sites. (A, B) H2A ChIP assays were performed on AsiSI-ER-U2OS cells treated with 4OHT or not for 4 h; the products were assessed by real-time q-PCR amplification using primers flanking the chr1_6:89231183 and chr6_12:90404906 DSB sites. Each value represents the mean ± S.D. of three independent experiments. (C, D) H2A ChIP assays were performed on AsiSI-ER-U2OS cells after treatment with 4OHT for 4 h or not; the products were assessed by real-time q-PCR amplification using primers near or distal to the chr22:19180307 DSB site. Each value represents the mean ± S.D. of three independent experiments.

Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification

Loss of BRG1 protects chromatin from MNase digestion. (A) U2OS cells were transfected with control or two BRG1 siRNAs (#3 and #5). Forty-eight hours later, the cells were lysed and detected by western blotting with BRG1 and actin antibodies. (B) U2OS cells were transfected with siRNA #3 for 48 h. Then, the cells were treated with 10 μM ETO or not for 20 min. One hour later, the nuclei were digested with 2 U, 5 U or 10 U MNase for 5 min at room temperature. The MNase-treated nucleosomal DNA was resolved on 1% agarose gels and visualized with ethidium bromide (upper panel). The quantification of the DNA amount marked by the white line is shown in the lower panel. siCont: control siRNA. siBRG1: BRG1 siRNA. Con: concentration.
Figure Legend Snippet: Loss of BRG1 protects chromatin from MNase digestion. (A) U2OS cells were transfected with control or two BRG1 siRNAs (#3 and #5). Forty-eight hours later, the cells were lysed and detected by western blotting with BRG1 and actin antibodies. (B) U2OS cells were transfected with siRNA #3 for 48 h. Then, the cells were treated with 10 μM ETO or not for 20 min. One hour later, the nuclei were digested with 2 U, 5 U or 10 U MNase for 5 min at room temperature. The MNase-treated nucleosomal DNA was resolved on 1% agarose gels and visualized with ethidium bromide (upper panel). The quantification of the DNA amount marked by the white line is shown in the lower panel. siCont: control siRNA. siBRG1: BRG1 siRNA. Con: concentration.

Techniques Used: Transfection, Western Blot, Concentration Assay

BRG1 depletion increases the percentage of apoptotic cells induced by ETO treatment. (A) SW13 cells were transfected with the pBJ5 empty vector or pBJ5-BRG1. Forty-eight hours later, the cells were treated with the indicated concentrations of ETO for 20 min, and apoptotic cells were detected by flow cytometry 24 h later. The percentage of apoptotic cells was analyzed by GraphPad 6. (B) U2OS cells were transfected with control siRNA or BRG1 siRNA for 48 h. Then, the cells were treated with the indicated concentrations of ETO for 20 min, and apoptotic cells were detected by flow cytometry 24 h later. The percentage of apoptotic cells was analyzed by GraphPad 6. The expression of BRG1 was analyzed by western blotting.
Figure Legend Snippet: BRG1 depletion increases the percentage of apoptotic cells induced by ETO treatment. (A) SW13 cells were transfected with the pBJ5 empty vector or pBJ5-BRG1. Forty-eight hours later, the cells were treated with the indicated concentrations of ETO for 20 min, and apoptotic cells were detected by flow cytometry 24 h later. The percentage of apoptotic cells was analyzed by GraphPad 6. (B) U2OS cells were transfected with control siRNA or BRG1 siRNA for 48 h. Then, the cells were treated with the indicated concentrations of ETO for 20 min, and apoptotic cells were detected by flow cytometry 24 h later. The percentage of apoptotic cells was analyzed by GraphPad 6. The expression of BRG1 was analyzed by western blotting.

Techniques Used: Transfection, Plasmid Preparation, Flow Cytometry, Cytometry, Expressing, Western Blot

BRG1 knockdown impairs DNA damage repair. (A) SW13 cells were transfected with the pBJ5 vector or pBJ5-BRG1 plasmids for 48 h. Then, the cells were treated with 10 μM ETO for 20 min and allowed to repair for the indicated time. The cells were fixed, permeabilized and immunostained with BRG1 and γ H2AX antibodies. Images were captured with a fluorescence microscope. The relative γ H2AX levels were quantified by ImageJ software. Scale bar: 10 μm. (B) U2OS cells were transfected with control siRNA or BRG1 siRNA for 48 h. Then, the cells were treated with 10 μM ETO for 20 min and allowed to repair for the indicated time. The cells were fixed, permeabilized and immunostained with BRG1 and γ H2AX antibodies. Images were captured with a fluorescence microscope. The relative γ H2AX levels were quantified by ImageJ software. Scale bar: 10 μm. siCont: control siRNA. siBRG1: BRG1 siRNA.
Figure Legend Snippet: BRG1 knockdown impairs DNA damage repair. (A) SW13 cells were transfected with the pBJ5 vector or pBJ5-BRG1 plasmids for 48 h. Then, the cells were treated with 10 μM ETO for 20 min and allowed to repair for the indicated time. The cells were fixed, permeabilized and immunostained with BRG1 and γ H2AX antibodies. Images were captured with a fluorescence microscope. The relative γ H2AX levels were quantified by ImageJ software. Scale bar: 10 μm. (B) U2OS cells were transfected with control siRNA or BRG1 siRNA for 48 h. Then, the cells were treated with 10 μM ETO for 20 min and allowed to repair for the indicated time. The cells were fixed, permeabilized and immunostained with BRG1 and γ H2AX antibodies. Images were captured with a fluorescence microscope. The relative γ H2AX levels were quantified by ImageJ software. Scale bar: 10 μm. siCont: control siRNA. siBRG1: BRG1 siRNA.

Techniques Used: Transfection, Plasmid Preparation, Fluorescence, Microscopy, Software

BRG1 depletion reduces chromatin relaxation near DSB sites. (A) U2OS cells expressing control siRNA or BRG1 siRNA #3 were incubated with 10 µM ETO. At the indicated repair times, the cell pellets were extracted in 1.0 M NaCl, and the released proteins were detected by western blotting analyses with the indicated antibodies. Ponceau S staining was used to demonstrate equal loading. (B) SW13 cells expressing HA-tagged wtBRG1, HA-tagged ATPase mutant BRG1 (K798R) or empty vector were treated with 10 μM ETO for 20 min and allowed to repair in fresh medium for 15, 30, 60 and 120 min. The cell pellets were extracted in 1.0 M NaCl, and the released histones were detected by western blotting analyses with the indicated antibodies. Ponceau S staining was used to demonstrate equal loading.
Figure Legend Snippet: BRG1 depletion reduces chromatin relaxation near DSB sites. (A) U2OS cells expressing control siRNA or BRG1 siRNA #3 were incubated with 10 µM ETO. At the indicated repair times, the cell pellets were extracted in 1.0 M NaCl, and the released proteins were detected by western blotting analyses with the indicated antibodies. Ponceau S staining was used to demonstrate equal loading. (B) SW13 cells expressing HA-tagged wtBRG1, HA-tagged ATPase mutant BRG1 (K798R) or empty vector were treated with 10 μM ETO for 20 min and allowed to repair in fresh medium for 15, 30, 60 and 120 min. The cell pellets were extracted in 1.0 M NaCl, and the released histones were detected by western blotting analyses with the indicated antibodies. Ponceau S staining was used to demonstrate equal loading.

Techniques Used: Expressing, Incubation, Western Blot, Staining, Mutagenesis, Plasmid Preparation

3) Product Images from "Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells"

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

Journal: Oncology Reports

doi: 10.3892/or.2014.3309

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

Techniques Used: Cell Culture, Staining, Transfection, MTT Assay

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

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

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

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

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

Techniques Used: Cell Culture, Staining, MTT Assay

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

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

4) Product Images from "Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells"

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

Journal: Oncology Reports

doi: 10.3892/or.2014.3309

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

Techniques Used: Cell Culture, Staining, Transfection, MTT Assay

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

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

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

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

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

Techniques Used: Cell Culture, Staining, MTT Assay

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

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

5) Product Images from "Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells"

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

Journal: Oncology Reports

doi: 10.3892/or.2014.3309

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

Techniques Used: Cell Culture, Staining, Transfection, MTT Assay

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

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

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

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

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

Techniques Used: Cell Culture, Staining, MTT Assay

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

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

6) Product Images from "RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells"

Article Title: RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells

Journal: Genome Biology

doi: 10.1186/s13059-016-0952-x

a Flow cytofluorimetry analysis of TNG-A Nanog::GFP cells at 4 DIV. b GFP-positive cell ratios at 2 DIV and 4 DIV after inhibition of SmarcA4 ( shSA4 ), DNMT ( AZA ), or KDM ( CHQ ) compared with control ( Ctrl ). c, d Similar analysis as in a and b , with a mESC line carrying GFP under the human Nanog promoter ( HNP ). e , f RT-PCR gene expression analysis. Values are relative to β-actin mRNA expression. The lowest and highest expression levels were normalized to 1 in the left and right histograms, respectively. g Nanog proximal promoter methylation. The scheme shows the region of the mouse Nanog promoter amplified for bisulfite-treated DNA sequencing (bis-seq). Black circles in grid rows indicate CpG methylation sites. The histogram shows the percentage of CpG methylation in different culture conditions as above. * p = 0.05, ** p = 0.01, *** p = 0.001 ( b , d , g , Student’s t -test; e , f , REST randomization test). Error bars show standard error
Figure Legend Snippet: a Flow cytofluorimetry analysis of TNG-A Nanog::GFP cells at 4 DIV. b GFP-positive cell ratios at 2 DIV and 4 DIV after inhibition of SmarcA4 ( shSA4 ), DNMT ( AZA ), or KDM ( CHQ ) compared with control ( Ctrl ). c, d Similar analysis as in a and b , with a mESC line carrying GFP under the human Nanog promoter ( HNP ). e , f RT-PCR gene expression analysis. Values are relative to β-actin mRNA expression. The lowest and highest expression levels were normalized to 1 in the left and right histograms, respectively. g Nanog proximal promoter methylation. The scheme shows the region of the mouse Nanog promoter amplified for bisulfite-treated DNA sequencing (bis-seq). Black circles in grid rows indicate CpG methylation sites. The histogram shows the percentage of CpG methylation in different culture conditions as above. * p = 0.05, ** p = 0.01, *** p = 0.001 ( b , d , g , Student’s t -test; e , f , REST randomization test). Error bars show standard error

Techniques Used: Flow Cytometry, Inhibition, Reverse Transcription Polymerase Chain Reaction, Expressing, Methylation, Amplification, DNA Sequencing, CpG Methylation Assay

a CRISPR/Cas9 guide design: the single guide RNA (sgRNA; red square ) was chosen to target a genomic region which is conserved between Ago1 and Ago2 but avoiding off-targets. b Western blot of Ago* proteins in control ( Ctrl ) or Ago1–2 CRISPR ES cells 4 days after transduction. c Western blot of Dnmt3b, SmarcA4, Kdm2b, Nanog, and GAPDH protein levels in ES cells cultured in 2i medium 4 days after transduction with Ago1–2 CRISPR lentiviral vector compared with control cells transduced with non-targeting CRISPR vector ( Ctrl CRISPR ). d mRNA levels of Dnmt3b, SmarcA4, and Kdm2b of cells as in c . e , f mRNA levels of pluripotency ( e ) or early neural commitment ( f ) in cells as in c and d 8 days after transduction. g , h GFP immunodetection in a 46C Sox1::GFP mESC line cultured in 2i medium 8 days after transduction with Ctrl CRISPR ( g ) or Ago1–2 CRISPR lentiviral vector ( h ). Scale bars , 50 microns. i , j Cell count ( i ) and cytofluorimetric analysis ( j ) of GFP-positive cells as in g and h . * p = 0.05, ** p = 0.01, *** p = 0.001 ( e – f , REST randomization test; i , Student’s t -test). Error bars show standard error
Figure Legend Snippet: a CRISPR/Cas9 guide design: the single guide RNA (sgRNA; red square ) was chosen to target a genomic region which is conserved between Ago1 and Ago2 but avoiding off-targets. b Western blot of Ago* proteins in control ( Ctrl ) or Ago1–2 CRISPR ES cells 4 days after transduction. c Western blot of Dnmt3b, SmarcA4, Kdm2b, Nanog, and GAPDH protein levels in ES cells cultured in 2i medium 4 days after transduction with Ago1–2 CRISPR lentiviral vector compared with control cells transduced with non-targeting CRISPR vector ( Ctrl CRISPR ). d mRNA levels of Dnmt3b, SmarcA4, and Kdm2b of cells as in c . e , f mRNA levels of pluripotency ( e ) or early neural commitment ( f ) in cells as in c and d 8 days after transduction. g , h GFP immunodetection in a 46C Sox1::GFP mESC line cultured in 2i medium 8 days after transduction with Ctrl CRISPR ( g ) or Ago1–2 CRISPR lentiviral vector ( h ). Scale bars , 50 microns. i , j Cell count ( i ) and cytofluorimetric analysis ( j ) of GFP-positive cells as in g and h . * p = 0.05, ** p = 0.01, *** p = 0.001 ( e – f , REST randomization test; i , Student’s t -test). Error bars show standard error

Techniques Used: CRISPR, Western Blot, Transduction, Cell Culture, Plasmid Preparation, Immunodetection, Cell Counting

7) Product Images from "DNA demethylation induces SALL4 gene re-expression in subgroups of hepatocellular carcinoma associated with Hepatitis B or C virus infection"

Article Title: DNA demethylation induces SALL4 gene re-expression in subgroups of hepatocellular carcinoma associated with Hepatitis B or C virus infection

Journal: Oncogene

doi: 10.1038/onc.2016.399

BRG1/BAF complex is involved in SALL4 re-expression in HBV replicating hepatocytes ( a, b ) PCR quantification of SALL4 mRNA (Left panel), and immunoblots of SALL4 (Right panel), following transfection of BRG1 siRNA (siBRG1) ( a ), and BRG1 plasmid ( b ), in HepAD38 cells grown with HBV replication by tetracycline removal for 10 days. Results are from three independent RNA isolations performed in identical triplicates. Error bars represent S.D. * P
Figure Legend Snippet: BRG1/BAF complex is involved in SALL4 re-expression in HBV replicating hepatocytes ( a, b ) PCR quantification of SALL4 mRNA (Left panel), and immunoblots of SALL4 (Right panel), following transfection of BRG1 siRNA (siBRG1) ( a ), and BRG1 plasmid ( b ), in HepAD38 cells grown with HBV replication by tetracycline removal for 10 days. Results are from three independent RNA isolations performed in identical triplicates. Error bars represent S.D. * P

Techniques Used: Expressing, Polymerase Chain Reaction, Western Blot, Transfection, Plasmid Preparation

HBV replication enables binding and interaction of STAT3, OCT4 and BRG1 in SALL4 regulatory region ( a ) ChIP assays with STAT3, OCT4, and BRG1 antibodies, as indicated, employing HepAD38 cells grown −/+ HBV replication by tetracycline removal for D0 and D10, and SALL4 primers spanning CpG sites shown in Fig.1b . Data were quantified as % of input. ( b ) Co-immunoprecipitations (co-IPs) of STAT3, OCT4 and BRG1 using indicated antibodies, employing extracts from HepAD38 cells grown −/+ HBV replication by tetracycline removal for D0 and D10. A representative assay is shown from three independent experiments. ( c ) Sequential ChIP assays of HepAD38 cells −/+ HBV replication by tetracycline removal for D0 and D10, using BRG1 antibody followed by tandem IP with STAT3 or OCT4 antibody. Results are from three independent experiments. Data were quantified as % of input.
Figure Legend Snippet: HBV replication enables binding and interaction of STAT3, OCT4 and BRG1 in SALL4 regulatory region ( a ) ChIP assays with STAT3, OCT4, and BRG1 antibodies, as indicated, employing HepAD38 cells grown −/+ HBV replication by tetracycline removal for D0 and D10, and SALL4 primers spanning CpG sites shown in Fig.1b . Data were quantified as % of input. ( b ) Co-immunoprecipitations (co-IPs) of STAT3, OCT4 and BRG1 using indicated antibodies, employing extracts from HepAD38 cells grown −/+ HBV replication by tetracycline removal for D0 and D10. A representative assay is shown from three independent experiments. ( c ) Sequential ChIP assays of HepAD38 cells −/+ HBV replication by tetracycline removal for D0 and D10, using BRG1 antibody followed by tandem IP with STAT3 or OCT4 antibody. Results are from three independent experiments. Data were quantified as % of input.

Techniques Used: Binding Assay, Chromatin Immunoprecipitation

8) Product Images from "SWI/SNF Mediates Polycomb Eviction and Epigenetic Reprogramming of the INK4b-ARF-INK4a Locus "

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

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.02019-07

hSNF5 mediates BRG1 recruitment to the p15 INK4b and p16 INK4a promoters. (A) ChIP-qPCR analysis of hSNF5 binding to the INK4b-ARF-INK4a locus revealed that hSNF5 binds directly to the p15 INK4b and p16 INKa promoters, but not to p14 ARF . Cross-linked chromatin was isolated from MRT cells that either lack (light green bars) or express (dark green bars) hSNF5. qPCR primer sets correspond to the p15 INK4b promoter (A), the p14 ARF promoter (B), an intergenic control region (C), and various regions of the p16 INK4a locus (sets D to I). Primer sets E and F cover the p16 INK4a promoter. The positions of the amplified regions on the INK4b-ARF-INK4a locus are indicated at the bottom. (B) BRG-1 binding to the p15 INK4b and p16 INK4a promoters is hSNF5 dependent, as revealed by ChIP-qPCR with antibodies directed against BRG-1. The procedures were as described in the legend to Fig. .
Figure Legend Snippet: hSNF5 mediates BRG1 recruitment to the p15 INK4b and p16 INK4a promoters. (A) ChIP-qPCR analysis of hSNF5 binding to the INK4b-ARF-INK4a locus revealed that hSNF5 binds directly to the p15 INK4b and p16 INKa promoters, but not to p14 ARF . Cross-linked chromatin was isolated from MRT cells that either lack (light green bars) or express (dark green bars) hSNF5. qPCR primer sets correspond to the p15 INK4b promoter (A), the p14 ARF promoter (B), an intergenic control region (C), and various regions of the p16 INK4a locus (sets D to I). Primer sets E and F cover the p16 INK4a promoter. The positions of the amplified regions on the INK4b-ARF-INK4a locus are indicated at the bottom. (B) BRG-1 binding to the p15 INK4b and p16 INK4a promoters is hSNF5 dependent, as revealed by ChIP-qPCR with antibodies directed against BRG-1. The procedures were as described in the legend to Fig. .

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

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.
Figure Legend 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.

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

9) Product Images from "SWI/SNF Mediates Polycomb Eviction and Epigenetic Reprogramming of the INK4b-ARF-INK4a Locus "

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

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.02019-07

hSNF5 mediates BRG1 recruitment to the p15 INK4b and p16 INK4a promoters. (A) ChIP-qPCR analysis of hSNF5 binding to the INK4b-ARF-INK4a locus revealed that hSNF5 binds directly to the p15 INK4b and p16 INKa promoters, but not to p14 ARF . Cross-linked chromatin was isolated from MRT cells that either lack (light green bars) or express (dark green bars) hSNF5. qPCR primer sets correspond to the p15 INK4b promoter (A), the p14 ARF promoter (B), an intergenic control region (C), and various regions of the p16 INK4a locus (sets D to I). Primer sets E and F cover the p16 INK4a promoter. The positions of the amplified regions on the INK4b-ARF-INK4a locus are indicated at the bottom. (B) BRG-1 binding to the p15 INK4b and p16 INK4a promoters is hSNF5 dependent, as revealed by ChIP-qPCR with antibodies directed against BRG-1. The procedures were as described in the legend to Fig. .
Figure Legend Snippet: hSNF5 mediates BRG1 recruitment to the p15 INK4b and p16 INK4a promoters. (A) ChIP-qPCR analysis of hSNF5 binding to the INK4b-ARF-INK4a locus revealed that hSNF5 binds directly to the p15 INK4b and p16 INKa promoters, but not to p14 ARF . Cross-linked chromatin was isolated from MRT cells that either lack (light green bars) or express (dark green bars) hSNF5. qPCR primer sets correspond to the p15 INK4b promoter (A), the p14 ARF promoter (B), an intergenic control region (C), and various regions of the p16 INK4a locus (sets D to I). Primer sets E and F cover the p16 INK4a promoter. The positions of the amplified regions on the INK4b-ARF-INK4a locus are indicated at the bottom. (B) BRG-1 binding to the p15 INK4b and p16 INK4a promoters is hSNF5 dependent, as revealed by ChIP-qPCR with antibodies directed against BRG-1. The procedures were as described in the legend to Fig. .

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

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.
Figure Legend 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.

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

10) Product Images from "BDNF rescues BAF53b-dependent synaptic plasticity and cocaine-associated memory in the nucleus accumbens"

Article Title: BDNF rescues BAF53b-dependent synaptic plasticity and cocaine-associated memory in the nucleus accumbens

Journal: Nature Communications

doi: 10.1038/ncomms11725

Characterization of Baf53b +/− heterozygous knockout and Baf53bΔHD mice. ( a ) nBAF complex is combinatorially assembled, meaning that the individual subunits included in each unique complex can alter CRC function 20 containing the dedicated and neuron-specific subunit BAF53b (highlighted in yellow). ( b ) WT Baf53b is illustrated with the hydrophobic domain (grey). Amino acids 323–333 in the hydrophobic domain were deleted to generate the BAF53bΔHD construct. This Baf53bΔHD mutant sequence was then cloned into a separate vector containing intron and exon sequences with splice sites and the SV40 intron and polyadenylation signal, which was then cloned downstream of the 8.5-kb mouse CamkIIα promoter 23 . This construct was then used to generate the Baf53bΔHD transgenic mice. ( c ) Immunoprecipitation and western blotting showing that Brg1 co-immunoprecipitates with BAF53b and BAF53bΔHD. ( d ) RT–qPCR revealed that the BAF53bΔHD transgene is only expressed in the BAF53bΔHD mutant mice and not in their WT littermates. Data are presented as mean±s.e.m. ( e ) RT–qPCR revealed that Baf53b +/− heterozygous knockout mice ( n =8) have significantly reduced WT Baf53b expression in the NAc compared with their WT littermates ( n =7; t (13)=10.25, P
Figure Legend Snippet: Characterization of Baf53b +/− heterozygous knockout and Baf53bΔHD mice. ( a ) nBAF complex is combinatorially assembled, meaning that the individual subunits included in each unique complex can alter CRC function 20 containing the dedicated and neuron-specific subunit BAF53b (highlighted in yellow). ( b ) WT Baf53b is illustrated with the hydrophobic domain (grey). Amino acids 323–333 in the hydrophobic domain were deleted to generate the BAF53bΔHD construct. This Baf53bΔHD mutant sequence was then cloned into a separate vector containing intron and exon sequences with splice sites and the SV40 intron and polyadenylation signal, which was then cloned downstream of the 8.5-kb mouse CamkIIα promoter 23 . This construct was then used to generate the Baf53bΔHD transgenic mice. ( c ) Immunoprecipitation and western blotting showing that Brg1 co-immunoprecipitates with BAF53b and BAF53bΔHD. ( d ) RT–qPCR revealed that the BAF53bΔHD transgene is only expressed in the BAF53bΔHD mutant mice and not in their WT littermates. Data are presented as mean±s.e.m. ( e ) RT–qPCR revealed that Baf53b +/− heterozygous knockout mice ( n =8) have significantly reduced WT Baf53b expression in the NAc compared with their WT littermates ( n =7; t (13)=10.25, P

Techniques Used: Knock-Out, Mouse Assay, Construct, Mutagenesis, Sequencing, Clone Assay, Plasmid Preparation, Transgenic Assay, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Expressing

11) Product Images from "LXR-Mediated ABCA1 Expression and Function Are Modulated by High Glucose and PRMT2"

Article Title: LXR-Mediated ABCA1 Expression and Function Are Modulated by High Glucose and PRMT2

Journal: PLoS ONE

doi: 10.1371/journal.pone.0135218

Chromatin-modifying enzymes expression and Abca1 CpG island methylation under high and normal glucose in macrophages. (A) RNA from RAW WT cells maintained under high (25 mM D-glucose) or normal (5.5 mM D-glucose + 19.5 mM L-glucose) glucose was profiled using a qRT-PCR-based chromatin-modifying array and expression changes in DNMT1 and PRMT2 are shown. (B) Epitect methyl CpG assay was employed to examine the methylation status of the Abca1 promoter CpG island. NIH 3T3 Mouse Genomic DNA and CpG Methylated NIH 3T3 Mouse Genomic DNA were used as controls. Input DNA was cleaved with methylation-sensitive and/or methylation-dependent restriction enzymes that digest unmethylated and methylated DNA, respectively. Following digestion, remaining DNA was quantified by qRT-PCR using primers that flank the CpG island. Percent methylation was determined by comparing the amount digested by each enzyme to a mock digested sample. (C) RNA from BMDMs differentiated under high (25 mM D-glucose) or normal (5.5 mM D-glucose + 19.5 mM L-glucose) glucose was extracted and levels of Prmt2 were profiled using qRT-PCR. (D) PRMT2 protein expression in normal and high glucose. Nuclear extract of BMDMs cultured as in C were blotted for PRMT2. Asterisk (*) represents a non-specific band, which serves as a loading control. (E) Nuclear protein extracts from BMDMs from wild type (WT) mice cultured in normal or high glucose and (F) whole cell extracts from BMDMs from wild type (WT) and Prmt2 -/- (KO) mice cultured under normal glucose were separated on a 4–20% gradient polyacrylamide gel and immunoblotted with an antibody specific for asymmetric dimethyl arginine. BRG1 or HSP90, serve as nuclear and whole-cell loading controls, respectively. Filled arrowheads represent asymmetric dimethyl arginine immunoreactive proteins uniquely present in one of the conditions.
Figure Legend Snippet: Chromatin-modifying enzymes expression and Abca1 CpG island methylation under high and normal glucose in macrophages. (A) RNA from RAW WT cells maintained under high (25 mM D-glucose) or normal (5.5 mM D-glucose + 19.5 mM L-glucose) glucose was profiled using a qRT-PCR-based chromatin-modifying array and expression changes in DNMT1 and PRMT2 are shown. (B) Epitect methyl CpG assay was employed to examine the methylation status of the Abca1 promoter CpG island. NIH 3T3 Mouse Genomic DNA and CpG Methylated NIH 3T3 Mouse Genomic DNA were used as controls. Input DNA was cleaved with methylation-sensitive and/or methylation-dependent restriction enzymes that digest unmethylated and methylated DNA, respectively. Following digestion, remaining DNA was quantified by qRT-PCR using primers that flank the CpG island. Percent methylation was determined by comparing the amount digested by each enzyme to a mock digested sample. (C) RNA from BMDMs differentiated under high (25 mM D-glucose) or normal (5.5 mM D-glucose + 19.5 mM L-glucose) glucose was extracted and levels of Prmt2 were profiled using qRT-PCR. (D) PRMT2 protein expression in normal and high glucose. Nuclear extract of BMDMs cultured as in C were blotted for PRMT2. Asterisk (*) represents a non-specific band, which serves as a loading control. (E) Nuclear protein extracts from BMDMs from wild type (WT) mice cultured in normal or high glucose and (F) whole cell extracts from BMDMs from wild type (WT) and Prmt2 -/- (KO) mice cultured under normal glucose were separated on a 4–20% gradient polyacrylamide gel and immunoblotted with an antibody specific for asymmetric dimethyl arginine. BRG1 or HSP90, serve as nuclear and whole-cell loading controls, respectively. Filled arrowheads represent asymmetric dimethyl arginine immunoreactive proteins uniquely present in one of the conditions.

Techniques Used: Expressing, Methylation, Quantitative RT-PCR, CpG Assay, Cell Culture, Mouse Assay, Gene Knockout

12) Product Images from "The long non-coding RNA Dali is an epigenetic regulator of neural differentiation"

Article Title: The long non-coding RNA Dali is an epigenetic regulator of neural differentiation

Journal: eLife

doi: 10.7554/eLife.04530

Dali associates with chromatin and transcriptional regulatory proteins. Dali interacts with BRG1, SIN3A, and P66beta in mouse N2A cells ( A ) and DNMT1 in mouse N2A and human SH-SY5Y cells ( B ). Nuclear extracts prepared from UV cross-linked cells were immuno-precipitated using either anti-DNMT1 or control IgG antibodies. Associated RNAs were purified and the levels of Dali and control Gapdh mRNA were quantified using qRT-PCR. Results are expressed as fold enrichment relative to an isotype IgG control antibody. Mean value ± s.e., n = 3. ( C ) De novo discovery of a near-perfect match to a CTCF motif in 125/1427 (8.8%) Dali CHART-Seq peaks. ( D ) Dali co-occupies several locations shared with CTCF. Control regions are not predicted to be bound by CTCF and are not bound by Dali . ChIP assays were performed in N2A cells using either an antibody against CTCF or an isotype specific control. The indicated DNA fragments were amplified using qPCR. Fold enrichment was calculated as 2-ΔΔCt (IP/IgG). Mean value ± s.e., n = 3. ( E ) Dali does not directly interact with CTCF protein in mouse N2A cells. Nuclear extracts were prepared from UV cross-linked cells and immuno-precipitated using either anti-CTCF or control IgG antibodies. Associated RNAs were purified and the levels of Dali and control U1 snoRNA were detected in each UV-RIP using qRT-PCR. Results are expressed as fold enrichment relative to an isotype IgG control antibody. Results are presented as mean value ± s.e. of three independent experiments. ( F ) De novo discovery of a motif for POU III family transcription factors (which includes POU3F3) in 115/1427 (8.1%) Dali CHART-Seq peaks. ( G ) UV-RIP in N2A cells: FLAG-tagged POU3F3 protein directly interacts with Dali . Mean value ± s.e., n = 3. ( H ) ChIP-qPCR in N2A cells: POU3F3 occupies a subset of loci bound by Dali and regulated by both Pou3f3 and Dali . Loci associated with known ( Dali -independent) Pou3f3 targets were used as positive control, while loci not regulated by either Pou3f3 or Dali and not bound by Dali were used as negative control. Mean value ± s.e., n = 3. DOI: http://dx.doi.org/10.7554/eLife.04530.013
Figure Legend Snippet: Dali associates with chromatin and transcriptional regulatory proteins. Dali interacts with BRG1, SIN3A, and P66beta in mouse N2A cells ( A ) and DNMT1 in mouse N2A and human SH-SY5Y cells ( B ). Nuclear extracts prepared from UV cross-linked cells were immuno-precipitated using either anti-DNMT1 or control IgG antibodies. Associated RNAs were purified and the levels of Dali and control Gapdh mRNA were quantified using qRT-PCR. Results are expressed as fold enrichment relative to an isotype IgG control antibody. Mean value ± s.e., n = 3. ( C ) De novo discovery of a near-perfect match to a CTCF motif in 125/1427 (8.8%) Dali CHART-Seq peaks. ( D ) Dali co-occupies several locations shared with CTCF. Control regions are not predicted to be bound by CTCF and are not bound by Dali . ChIP assays were performed in N2A cells using either an antibody against CTCF or an isotype specific control. The indicated DNA fragments were amplified using qPCR. Fold enrichment was calculated as 2-ΔΔCt (IP/IgG). Mean value ± s.e., n = 3. ( E ) Dali does not directly interact with CTCF protein in mouse N2A cells. Nuclear extracts were prepared from UV cross-linked cells and immuno-precipitated using either anti-CTCF or control IgG antibodies. Associated RNAs were purified and the levels of Dali and control U1 snoRNA were detected in each UV-RIP using qRT-PCR. Results are expressed as fold enrichment relative to an isotype IgG control antibody. Results are presented as mean value ± s.e. of three independent experiments. ( F ) De novo discovery of a motif for POU III family transcription factors (which includes POU3F3) in 115/1427 (8.1%) Dali CHART-Seq peaks. ( G ) UV-RIP in N2A cells: FLAG-tagged POU3F3 protein directly interacts with Dali . Mean value ± s.e., n = 3. ( H ) ChIP-qPCR in N2A cells: POU3F3 occupies a subset of loci bound by Dali and regulated by both Pou3f3 and Dali . Loci associated with known ( Dali -independent) Pou3f3 targets were used as positive control, while loci not regulated by either Pou3f3 or Dali and not bound by Dali were used as negative control. Mean value ± s.e., n = 3. DOI: http://dx.doi.org/10.7554/eLife.04530.013

Techniques Used: Purification, Quantitative RT-PCR, Chromatin Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Positive Control, Negative Control

13) Product Images from "The dilemma of early preventive oophorectomy in familial small cell carcinoma of the ovary of hypercalcemic type"

Article Title: The dilemma of early preventive oophorectomy in familial small cell carcinoma of the ovary of hypercalcemic type

Journal: Gynecologic Oncology Reports

doi: 10.1016/j.gore.2019.02.002

Retained SMARCA4 (BRG1) protein expression in one of the ovaries. There is positive nuclear staining of the granulosa cells of a developing follicle (right of photomicrograph) and the ovarian stroma (left of photomicrograph).
Figure Legend Snippet: Retained SMARCA4 (BRG1) protein expression in one of the ovaries. There is positive nuclear staining of the granulosa cells of a developing follicle (right of photomicrograph) and the ovarian stroma (left of photomicrograph).

Techniques Used: Expressing, Staining

14) Product Images from "MeCP2 interacts with chromosomal microRNAs in brain"

Article Title: MeCP2 interacts with chromosomal microRNAs in brain

Journal: Epigenetics

doi: 10.1080/15592294.2017.1391429

RNA immunoprecipitation sequencing (RIP-seq) analysis. (A) Illustration of native RNA immunoprecipitation combined with massive parallel sequencing (nRIP-seq) protocol. (B-C) Micrococcal nuclease digestion of mouse primary cortical neurons. (B) Gel electrophoresis of DNA purified from different fractions in mouse primary cortical neurons after MNase digestion for 12 minutes. S1 is MNase sensitive chromatin fraction contains mainly mononucleosomes whereas S2 is MNase resistant chromatin fraction containing nucleosomal ladder (Di, Tri and so on). (C) Western blot analysis of MNase digested chromatin fractions in mice cortical neurons with antibodies against MeCP2, Hp1α, Brg1, Brm, Dhx9, and Pol II. (D) Smear plots showing log2 fold change (log2FC) plotted against log2 counts per million (CPM). Red dots on positive scale in plot represent small transcripts (125) with increased binding with MeCP2 in MNase resistant S2 chromatin fraction relative to MNase sensitive S1 chromatin fraction. (E) Percentage of different classes of RNA transcripts bound with MeCP2 where microRNAs were the most abundant small ncRNAs that were bound by MeCP2.
Figure Legend Snippet: RNA immunoprecipitation sequencing (RIP-seq) analysis. (A) Illustration of native RNA immunoprecipitation combined with massive parallel sequencing (nRIP-seq) protocol. (B-C) Micrococcal nuclease digestion of mouse primary cortical neurons. (B) Gel electrophoresis of DNA purified from different fractions in mouse primary cortical neurons after MNase digestion for 12 minutes. S1 is MNase sensitive chromatin fraction contains mainly mononucleosomes whereas S2 is MNase resistant chromatin fraction containing nucleosomal ladder (Di, Tri and so on). (C) Western blot analysis of MNase digested chromatin fractions in mice cortical neurons with antibodies against MeCP2, Hp1α, Brg1, Brm, Dhx9, and Pol II. (D) Smear plots showing log2 fold change (log2FC) plotted against log2 counts per million (CPM). Red dots on positive scale in plot represent small transcripts (125) with increased binding with MeCP2 in MNase resistant S2 chromatin fraction relative to MNase sensitive S1 chromatin fraction. (E) Percentage of different classes of RNA transcripts bound with MeCP2 where microRNAs were the most abundant small ncRNAs that were bound by MeCP2.

Techniques Used: Immunoprecipitation, Sequencing, Nucleic Acid Electrophoresis, Purification, Western Blot, Mouse Assay, Binding Assay

15) Product Images from "Involvement of Telomerase Reverse Transcriptase in Heterochromatin Maintenance"

Article Title: Involvement of Telomerase Reverse Transcriptase in Heterochromatin Maintenance

Journal:

doi: 10.1128/MCB.00093-14

hTERT, BRG1, and NS form a functional complex in mitotic cells. (A) Telomerase activity of HeLa cells with or without manipulation. Telomerase activity was detected by TRAP assay. IC, internal control. (B) Immunoblot (IB) using the indicated antibodies
Figure Legend Snippet: hTERT, BRG1, and NS form a functional complex in mitotic cells. (A) Telomerase activity of HeLa cells with or without manipulation. Telomerase activity was detected by TRAP assay. IC, internal control. (B) Immunoblot (IB) using the indicated antibodies

Techniques Used: Functional Assay, Activity Assay, TRAP Assay

16) Product Images from "The spring-loaded genome: Nucleosome redistributions are widespread, transient, and DNA-directed"

Article Title: The spring-loaded genome: Nucleosome redistributions are widespread, transient, and DNA-directed

Journal:

doi: 10.1101/gr.160150.113

Nucleosome redistributions are determined by the underlying DNA sequence. ( A ) Western blots with the specified antibodies, at various times (in hours) after KSHV reactivation (hpr), of iSLK.219 cells treated with 0.2 µg/mL doxycycline. BRG1 protein
Figure Legend Snippet: Nucleosome redistributions are determined by the underlying DNA sequence. ( A ) Western blots with the specified antibodies, at various times (in hours) after KSHV reactivation (hpr), of iSLK.219 cells treated with 0.2 µg/mL doxycycline. BRG1 protein

Techniques Used: Sequencing, Western Blot

17) Product Images from "SNF5 Is an Essential Executor of Epigenetic Regulation during Differentiation"

Article Title: SNF5 Is an Essential Executor of Epigenetic Regulation during Differentiation

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003459

SNF5 is recruited to OCT4-activated and -repressed genes with distinctive chromatin landscape during differentiation. (A–E) Chromatin from NCCIT cells was immunoprecipitated with anti-OCT4 (A), anti-EZH2 (B), anti-SNF5 (C), anti-BRM (D), anti-BRG1 (E) or anti-H3 antibodies and their binding at the DNA regulatory regions of OCT4 target genes were analyzed by quantitative PCR. Quantitative PCR data represent the average of three biological experiments (the mean +SEM). A Mann-Whitney test was performed and the increase in recruitment of SNF5, BRM and BRG1 at OCT4 target genes during differentiation as found to be statistically significant with p-values of 0.013 (SNF5), 0.012 (BRM) and 0.029 (BRG1). (F) The protein level of EZH2, SNF5, BRG1, BRM and loading control ACTIN were subsequently analyzed by western blot. The data is representative of three biological experiments.
Figure Legend Snippet: SNF5 is recruited to OCT4-activated and -repressed genes with distinctive chromatin landscape during differentiation. (A–E) Chromatin from NCCIT cells was immunoprecipitated with anti-OCT4 (A), anti-EZH2 (B), anti-SNF5 (C), anti-BRM (D), anti-BRG1 (E) or anti-H3 antibodies and their binding at the DNA regulatory regions of OCT4 target genes were analyzed by quantitative PCR. Quantitative PCR data represent the average of three biological experiments (the mean +SEM). A Mann-Whitney test was performed and the increase in recruitment of SNF5, BRM and BRG1 at OCT4 target genes during differentiation as found to be statistically significant with p-values of 0.013 (SNF5), 0.012 (BRM) and 0.029 (BRG1). (F) The protein level of EZH2, SNF5, BRG1, BRM and loading control ACTIN were subsequently analyzed by western blot. The data is representative of three biological experiments.

Techniques Used: Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Western Blot

18) Product Images from "Transient bursts of Zscan4 expression are accompanied by the rapid derepression of heterochromatin in mouse embryonic stem cells"

Article Title: Transient bursts of Zscan4 expression are accompanied by the rapid derepression of heterochromatin in mouse embryonic stem cells

Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

doi: 10.1093/dnares/dsv013

Both activating and repressing chromatin remodelling complexes localize in heterochromatin in the Zscan4 + cells. (A) Immunoblot analyses of Flag-Zscan4, Lsd1/Kdm1a, Mta1, Brg1, Hdac1, and Kap1/Trim28 proteins after immunoprecipitating the nuclear extracts of tet-Zscan4 ES cells by antibodies against Flag-tag, Lsd1/Kdm1a, Mta1, and Brg1. The tet-Zscan4 ES cells were cultured in the Dox + (without Zscan4 overexpression) and Dox − (with Zscan4 overexpression) for 3 days. *, possible cross-reactive polypeptides. **, non-specific bands. (B) Triple immunostaining analyses of ES cells with the Zscan4 antibody (red), the CREST antibody (white, an anti-centromere protein), and various antibodies indicated (green). Blue, DAPI. Arrows indicate the clustered centromeres. Scale bars, 5 µm. See also Supplementary Fig. S5 .
Figure Legend Snippet: Both activating and repressing chromatin remodelling complexes localize in heterochromatin in the Zscan4 + cells. (A) Immunoblot analyses of Flag-Zscan4, Lsd1/Kdm1a, Mta1, Brg1, Hdac1, and Kap1/Trim28 proteins after immunoprecipitating the nuclear extracts of tet-Zscan4 ES cells by antibodies against Flag-tag, Lsd1/Kdm1a, Mta1, and Brg1. The tet-Zscan4 ES cells were cultured in the Dox + (without Zscan4 overexpression) and Dox − (with Zscan4 overexpression) for 3 days. *, possible cross-reactive polypeptides. **, non-specific bands. (B) Triple immunostaining analyses of ES cells with the Zscan4 antibody (red), the CREST antibody (white, an anti-centromere protein), and various antibodies indicated (green). Blue, DAPI. Arrows indicate the clustered centromeres. Scale bars, 5 µm. See also Supplementary Fig. S5 .

Techniques Used: FLAG-tag, Cell Culture, Over Expression, Triple Immunostaining

Related Articles

Transduction:

Article Title: SWI/SNF Mediates Polycomb Eviction and Epigenetic Reprogramming of the INK4b-ARF-INK4a Locus
Article Snippet: Cross-linked chromatin was prepared from ∼2 × 107 cells 48 h after transduction with lentiviruses expressing either hSNF5 or GFP as a control. .. The following antibodies were used: SNF5 (ab12167 [Abcam]), BRG1 (ab4081 [Abcam]), SUZ12 (ab12073 [Abcam]), BMI1 (ab14389 [Abcam]), EZH2 (Sc-25383 [Santa Cruz]), DNMT3B (IMG-184A [IMGENEX]), POL II (Sc-899 [Santa Cruz]), mixed lineage leukemia 1 (MLL1; A300-086A [Bethyl Laboratories]), Histone H3 (ab1791 [Abcam]), H3-K4me3 (ab12209 [Abcam]), and H3-K27me3 (catalog no. 07-449 [Upstate]).

Clone Assay:

Article Title: BDNF rescues BAF53b-dependent synaptic plasticity and cocaine-associated memory in the nucleus accumbens
Article Snippet: WT Baf53b and Baf53bΔHD were cloned into pLVX-EF1a-IRES-mCherry (Clontech, Mountain View, CA) creating plasmids MW95 and MW97, respectively. .. Immunoprecipitations against Brg1 were performed using 1 μg of antibody ab70558 (Abcam,, Cambridge, MA).

Centrifugation:

Article Title: RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells
Article Snippet: Supernatant was harvested by centrifugation (10 min at 13,000 RPM, 4 °C) and quantified with a Micro BCA Protein Assay Kit (Thermo Scientific). .. The total protein extract (10 to 50 μg) was resolved on 8–10 % acrylamide gels, transferred on a nitrocellulose membrane (Hybond-c Extra, GE Healthcare), blocked with 5 % milk proteins in TBST (50 mM Tris pH 7.6, 150 mM NaCl, 0.05 % Tween-20), and probed with primary antibodies, including Dnmt3b (1:2000; Imgenex/Novus Biologicals; IMG-184A), SmarcA4 (1:1000; Abcam; ab4081), Kdm2b (1:2000; Merck Millipore; 09-864), PolII (clone CTD4H8; 1:1000; Millipore), α-tubulin (clone B-5-1-2; 1:5000; Sigma-Aldrich; T6074), GAPDH (Sigma-Aldrich; G9545), panAgo clone 2A8(1:250; Millipore; MABE56) and Ago2 clone 9E8.2 (1:2000; Millipore; 04-642).

Article Title: LXR-Mediated ABCA1 Expression and Function Are Modulated by High Glucose and PRMT2
Article Snippet: Cellular debris was pelleted by centrifugation at 16,000 x g for 15 minutes at 4 o C. Supernatant was collected and protein concentrations were determined by the Bradford assay (Biorad). .. Antibodies used were anti-ABCA1 (1:1,000, Novus 400–105), anti-HSP90 (1:500, BD 610419), anti-LXRα (1:1,000, Abcam ab41902), anti-Myc (1:2,000, Cell Signaling 2276), and anti-dimethyl-arginine, asymmetric (ASYM25) (1:1,000, EMD Millipore 07–414), and anti-BRG1 (1:1,000, Abcam ab4081).

Article Title: Involvement of Telomerase Reverse Transcriptase in Heterochromatin Maintenance
Article Snippet: The lysate was preabsorbed with 40 μl of Pierce protein A plus agarose (Thermo Scientific) for 30 min. Preabsorbed lysate was mixed with 10 μg of anti-hTERT MAb (clone 10E9-2), anti-BRG1 antibody (ab4081; Abcam), or anti-NS antibody (A300-599A; Bethyl Laboratories) and 40 μl of Pierce protein A plus agarose and incubated overnight at 4°C. .. The lysate was preabsorbed with 40 μl of Pierce protein A plus agarose (Thermo Scientific) for 30 min. Preabsorbed lysate was mixed with 10 μg of anti-hTERT MAb (clone 10E9-2), anti-BRG1 antibody (ab4081; Abcam), or anti-NS antibody (A300-599A; Bethyl Laboratories) and 40 μl of Pierce protein A plus agarose and incubated overnight at 4°C.

Amplification:

Article Title: Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells
Article Snippet: ChIP was performed overnight at 4°C using the BRG1 antibody (ab4081; Abcam) or an isotype control IgG. .. After a 1 h incubation in the presence of salmon sperm DNA/protein A agarose beads, the immunoprecipitated DNA/protein complexes were then washed and eluted from the beads with 1% SDS and 0.1 M NaHCO3 solution.

Bradford Assay:

Article Title: LXR-Mediated ABCA1 Expression and Function Are Modulated by High Glucose and PRMT2
Article Snippet: Cellular debris was pelleted by centrifugation at 16,000 x g for 15 minutes at 4 o C. Supernatant was collected and protein concentrations were determined by the Bradford assay (Biorad). .. Antibodies used were anti-ABCA1 (1:1,000, Novus 400–105), anti-HSP90 (1:500, BD 610419), anti-LXRα (1:1,000, Abcam ab41902), anti-Myc (1:2,000, Cell Signaling 2276), and anti-dimethyl-arginine, asymmetric (ASYM25) (1:1,000, EMD Millipore 07–414), and anti-BRG1 (1:1,000, Abcam ab4081).

Blocking Assay:

Article Title: RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells
Article Snippet: The total protein extract (10 to 50 μg) was resolved on 8–10 % acrylamide gels, transferred on a nitrocellulose membrane (Hybond-c Extra, GE Healthcare), blocked with 5 % milk proteins in TBST (50 mM Tris pH 7.6, 150 mM NaCl, 0.05 % Tween-20), and probed with primary antibodies, including Dnmt3b (1:2000; Imgenex/Novus Biologicals; IMG-184A), SmarcA4 (1:1000; Abcam; ab4081), Kdm2b (1:2000; Merck Millipore; 09-864), PolII (clone CTD4H8; 1:1000; Millipore), α-tubulin (clone B-5-1-2; 1:5000; Sigma-Aldrich; T6074), GAPDH (Sigma-Aldrich; G9545), panAgo clone 2A8(1:250; Millipore; MABE56) and Ago2 clone 9E8.2 (1:2000; Millipore; 04-642). .. The total protein extract (10 to 50 μg) was resolved on 8–10 % acrylamide gels, transferred on a nitrocellulose membrane (Hybond-c Extra, GE Healthcare), blocked with 5 % milk proteins in TBST (50 mM Tris pH 7.6, 150 mM NaCl, 0.05 % Tween-20), and probed with primary antibodies, including Dnmt3b (1:2000; Imgenex/Novus Biologicals; IMG-184A), SmarcA4 (1:1000; Abcam; ab4081), Kdm2b (1:2000; Merck Millipore; 09-864), PolII (clone CTD4H8; 1:1000; Millipore), α-tubulin (clone B-5-1-2; 1:5000; Sigma-Aldrich; T6074), GAPDH (Sigma-Aldrich; G9545), panAgo clone 2A8(1:250; Millipore; MABE56) and Ago2 clone 9E8.2 (1:2000; Millipore; 04-642).

Article Title: MeCP2 interacts with chromosomal microRNAs in brain
Article Snippet: PVDF membranes were incubated in blocking buffer (3% BSA in PBST (PBS containing 0.05% v/v tween 20) for 1 h at RT. .. Antibodies used were anti-Brm (Abcam, ab15597), anti-Brg1 (Abcam, ab4081), anti-Dhx9 (ThermoFisher, PA5-19542), anti-Hp1 alpha (Abcam, ab77256), anti-MeCP2 antibody (M9317, Sigma), anti-Pol II (Millipore, 05-623).

Article Title: Bmi1 and BRG1 drive myocardial repair by regulating cardiac stem cell function in acute rheumatic heart disease
Article Snippet: Non-specific sites were blocked using 4% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) solution at room temperature for 1 h. Subsequently, the sections were treated with primary antibodies (1:400) directed against c-kit (cat. no. ab32363), Bmi1 (cat. no. ab38295) or BRG1 (cat. no. ab70558; all Abcam, Cambridge, UK) overnight at 4°C. .. Non-specific sites were blocked using 4% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) solution at room temperature for 1 h. Subsequently, the sections were treated with primary antibodies (1:400) directed against c-kit (cat. no. ab32363), Bmi1 (cat. no. ab38295) or BRG1 (cat. no. ab70558; all Abcam, Cambridge, UK) overnight at 4°C.

Real-time Polymerase Chain Reaction:

Article Title: Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells
Article Snippet: Next, we investigated UCA1 and BRG1 binding in vivo by RNA-binding protein immunoprecipitation (RIP) experiments. .. BRG1 antibody was used to immunoprecipitate BRG1 protein and associated RNAs in 5637 cells, and the presence of UCA1 RNA was determined by quantitative real-time PCR. .. Mouse IgG was used as control.

Incubation:

Article Title: RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells
Article Snippet: Cells were lysed with RIPA buffer (50 mM Tris-HCl pH 7.6, 1 % NP40, 0.5 % deoxycholic acid, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 % SDS) supplemented with Complete Protease Inhibitor Cocktail (Roche); lysate was incubated for 30 min on ice and sonicated three times for 10 sec each on medium power in order to reduce viscosity. .. The total protein extract (10 to 50 μg) was resolved on 8–10 % acrylamide gels, transferred on a nitrocellulose membrane (Hybond-c Extra, GE Healthcare), blocked with 5 % milk proteins in TBST (50 mM Tris pH 7.6, 150 mM NaCl, 0.05 % Tween-20), and probed with primary antibodies, including Dnmt3b (1:2000; Imgenex/Novus Biologicals; IMG-184A), SmarcA4 (1:1000; Abcam; ab4081), Kdm2b (1:2000; Merck Millipore; 09-864), PolII (clone CTD4H8; 1:1000; Millipore), α-tubulin (clone B-5-1-2; 1:5000; Sigma-Aldrich; T6074), GAPDH (Sigma-Aldrich; G9545), panAgo clone 2A8(1:250; Millipore; MABE56) and Ago2 clone 9E8.2 (1:2000; Millipore; 04-642).

Article Title: LXR-Mediated ABCA1 Expression and Function Are Modulated by High Glucose and PRMT2
Article Snippet: Membranes were blocked with 5% BSA in TBS for 1 hour and incubated in primary antibody (diluted in 5% BSA/TBS) at 4°C overnight. .. Antibodies used were anti-ABCA1 (1:1,000, Novus 400–105), anti-HSP90 (1:500, BD 610419), anti-LXRα (1:1,000, Abcam ab41902), anti-Myc (1:2,000, Cell Signaling 2276), and anti-dimethyl-arginine, asymmetric (ASYM25) (1:1,000, EMD Millipore 07–414), and anti-BRG1 (1:1,000, Abcam ab4081).

Article Title: MeCP2 interacts with chromosomal microRNAs in brain
Article Snippet: The membrane was incubated with an appropriate secondary horseradish peroxidase conjugated antibody (diluted 1:20,000 in wash buffer) for 30 min at RT with agitation followed by 4-5 washes in PBST buffer. .. Antibodies used were anti-Brm (Abcam, ab15597), anti-Brg1 (Abcam, ab4081), anti-Dhx9 (ThermoFisher, PA5-19542), anti-Hp1 alpha (Abcam, ab77256), anti-MeCP2 antibody (M9317, Sigma), anti-Pol II (Millipore, 05-623).

Article Title: Involvement of Telomerase Reverse Transcriptase in Heterochromatin Maintenance
Article Snippet: After sonication, lysates were cleared of insoluble material by centrifugation at 21,000 × g at 4°C for 15 min. .. The lysate was preabsorbed with 40 μl of Pierce protein A plus agarose (Thermo Scientific) for 30 min. Preabsorbed lysate was mixed with 10 μg of anti-hTERT MAb (clone 10E9-2), anti-BRG1 antibody (ab4081; Abcam), or anti-NS antibody (A300-599A; Bethyl Laboratories) and 40 μl of Pierce protein A plus agarose and incubated overnight at 4°C. .. Immune complexes were washed four times with 1× acetate buffer (10 mM HEPES-KOH [pH 7.8], 100 mM potassium acetate, and 4 mM MgCl2 ) containing 10% glycerol, 0.1% Triton X, and 0.06× protease inhibitor and once with AGC solution [1× acetate buffer, 10% glycerol, and 0.02% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) containing 2 mM CaCl2 .

Article Title: The spring-loaded genome: Nucleosome redistributions are widespread, transient, and DNA-directed
Article Snippet: The BRG1 antibody (#4081) was purchased from Abcam. .. Whole-cell extract equivalent to 0.1 × 106 cells was resolved on SDS-PAGE 10% Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes.

Activity Assay:

Article Title: Bmi1 and BRG1 drive myocardial repair by regulating cardiac stem cell function in acute rheumatic heart disease
Article Snippet: Endogenous peroxidase activity was blocked by treating with 10% H2 O2 for 30 min at room temperature. .. Non-specific sites were blocked using 4% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) solution at room temperature for 1 h. Subsequently, the sections were treated with primary antibodies (1:400) directed against c-kit (cat. no. ab32363), Bmi1 (cat. no. ab38295) or BRG1 (cat. no. ab70558; all Abcam, Cambridge, UK) overnight at 4°C.

Expressing:

Article Title: SWI/SNF Mediates Polycomb Eviction and Epigenetic Reprogramming of the INK4b-ARF-INK4a Locus
Article Snippet: Cross-linked chromatin was prepared from ∼2 × 107 cells 48 h after transduction with lentiviruses expressing either hSNF5 or GFP as a control. .. The following antibodies were used: SNF5 (ab12167 [Abcam]), BRG1 (ab4081 [Abcam]), SUZ12 (ab12073 [Abcam]), BMI1 (ab14389 [Abcam]), EZH2 (Sc-25383 [Santa Cruz]), DNMT3B (IMG-184A [IMGENEX]), POL II (Sc-899 [Santa Cruz]), mixed lineage leukemia 1 (MLL1; A300-086A [Bethyl Laboratories]), Histone H3 (ab1791 [Abcam]), H3-K4me3 (ab12209 [Abcam]), and H3-K27me3 (catalog no. 07-449 [Upstate]).

Article Title: MeCP2 interacts with chromosomal microRNAs in brain
Article Snippet: Protein expression was visualized using enhanced chemiluminescent reagent kit (Sigma-Aldrich) and detected by exposing membranes to high performance chemiluminescent film for an optimized time. .. Antibodies used were anti-Brm (Abcam, ab15597), anti-Brg1 (Abcam, ab4081), anti-Dhx9 (ThermoFisher, PA5-19542), anti-Hp1 alpha (Abcam, ab77256), anti-MeCP2 antibody (M9317, Sigma), anti-Pol II (Millipore, 05-623).

BIA-KA:

Article Title: RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells
Article Snippet: Supernatant was harvested by centrifugation (10 min at 13,000 RPM, 4 °C) and quantified with a Micro BCA Protein Assay Kit (Thermo Scientific). .. The total protein extract (10 to 50 μg) was resolved on 8–10 % acrylamide gels, transferred on a nitrocellulose membrane (Hybond-c Extra, GE Healthcare), blocked with 5 % milk proteins in TBST (50 mM Tris pH 7.6, 150 mM NaCl, 0.05 % Tween-20), and probed with primary antibodies, including Dnmt3b (1:2000; Imgenex/Novus Biologicals; IMG-184A), SmarcA4 (1:1000; Abcam; ab4081), Kdm2b (1:2000; Merck Millipore; 09-864), PolII (clone CTD4H8; 1:1000; Millipore), α-tubulin (clone B-5-1-2; 1:5000; Sigma-Aldrich; T6074), GAPDH (Sigma-Aldrich; G9545), panAgo clone 2A8(1:250; Millipore; MABE56) and Ago2 clone 9E8.2 (1:2000; Millipore; 04-642).

Modification:

Article Title: Involvement of Telomerase Reverse Transcriptase in Heterochromatin Maintenance
Article Snippet: IP followed by an RdRP (IP-RdRP) assay was modified from the original protocol ( ). .. The lysate was preabsorbed with 40 μl of Pierce protein A plus agarose (Thermo Scientific) for 30 min. Preabsorbed lysate was mixed with 10 μg of anti-hTERT MAb (clone 10E9-2), anti-BRG1 antibody (ab4081; Abcam), or anti-NS antibody (A300-599A; Bethyl Laboratories) and 40 μl of Pierce protein A plus agarose and incubated overnight at 4°C.

Western Blot:

Article Title: Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells
Article Snippet: Paragraph title: Western blot analysis ... Antibodies used for immunoblotting were anti-β-actin antibody (PM053; MBL, Japan) (1:5,000), anti-human p21 (3733-1; Abcam Epitomics, Cambridge, UK) (1:2,000), anti-H3K9me3 (49–1008; Novex, Carlsbad, CA, USA) (1:1,000), anti-H3K4m3 (ab8580) (1:2,000) and anti-human BRG1 antibodies (ab4081) (both from Abcam) (1:2,000).

Article Title: RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells
Article Snippet: Paragraph title: Western blotting ... The total protein extract (10 to 50 μg) was resolved on 8–10 % acrylamide gels, transferred on a nitrocellulose membrane (Hybond-c Extra, GE Healthcare), blocked with 5 % milk proteins in TBST (50 mM Tris pH 7.6, 150 mM NaCl, 0.05 % Tween-20), and probed with primary antibodies, including Dnmt3b (1:2000; Imgenex/Novus Biologicals; IMG-184A), SmarcA4 (1:1000; Abcam; ab4081), Kdm2b (1:2000; Merck Millipore; 09-864), PolII (clone CTD4H8; 1:1000; Millipore), α-tubulin (clone B-5-1-2; 1:5000; Sigma-Aldrich; T6074), GAPDH (Sigma-Aldrich; G9545), panAgo clone 2A8(1:250; Millipore; MABE56) and Ago2 clone 9E8.2 (1:2000; Millipore; 04-642).

Article Title: SWI/SNF Mediates Polycomb Eviction and Epigenetic Reprogramming of the INK4b-ARF-INK4a Locus
Article Snippet: Paragraph title: Cell extracts and Western blotting. ... Primary antibodies included SNF5 (ab12167 [Abcam]), BRG1 (ab4081 [Abcam]), SUZ12 (ab12073 [Abcam]), BMI1 (ab14389 [Abcam]), EZH2 (Sc-25383 [Santa Cruz]), DNMT3B (IMG-184A [IMGENEX]), and histone H3 (ab1791 [Abcam]).

Article Title: BDNF rescues BAF53b-dependent synaptic plasticity and cocaine-associated memory in the nucleus accumbens
Article Snippet: Paragraph title: Immunoprecipitation and western blotting ... Immunoprecipitations against Brg1 were performed using 1 μg of antibody ab70558 (Abcam,, Cambridge, MA).

Article Title: LXR-Mediated ABCA1 Expression and Function Are Modulated by High Glucose and PRMT2
Article Snippet: Paragraph title: Preparation of cell extracts and Western blotting ... Antibodies used were anti-ABCA1 (1:1,000, Novus 400–105), anti-HSP90 (1:500, BD 610419), anti-LXRα (1:1,000, Abcam ab41902), anti-Myc (1:2,000, Cell Signaling 2276), and anti-dimethyl-arginine, asymmetric (ASYM25) (1:1,000, EMD Millipore 07–414), and anti-BRG1 (1:1,000, Abcam ab4081).

Article Title: The spring-loaded genome: Nucleosome redistributions are widespread, transient, and DNA-directed
Article Snippet: Paragraph title: Antibodies and Western blotting ... The BRG1 antibody (#4081) was purchased from Abcam.

Article Title: SNF5 Is an Essential Executor of Epigenetic Regulation during Differentiation
Article Snippet: Paragraph title: Western blot analysis ... Antibodies against SNF5 (ab12167), BRG1 (ab4081), BRM (ab15597), and BAF53b (ab103771) were purchased from Abcam, Inc (Cambridge, MA).

Article Title: Transient bursts of Zscan4 expression are accompanied by the rapid derepression of heterochromatin in mouse embryonic stem cells
Article Snippet: The following antibodies were used: mouse polyclonal anti-Zscan4 (Abnova H00201516- B01P), rabbit polyclonal anti-Zscan4, HP1α (Millipore MAB3584 for immunostaining, Millipore no. 05-689 for immunoblotting), H3K9me3 (Upstate no. 07-442), H4K20me3 (Millipore no. 07-463), H3K4me3 (Millipore no. 07-473), H3K9ac (Millipore no. 07-352), H3K14ac (Millipore no. 17-10051), H3K18ac (Epitomics no. 1766-1), H3K27ac (Active Motif no. 39-133 for immunostaining and ChIP, Abcam ab4729 for immunostaining and immunoblotting), H3K9me2 (Abcam ab1220), pan-H3 (Abcam ab1791), p300 (Millipore no. 05-257), CBP (Santa Cruz sc-583), HDAC1 (Abcam ab7028 for immunostaining, ab31263 for western blot), HDAC2 (ab7029), MTA1 (Bethyl lab. .. A300-911A for immunostaining, Abcam ab71153 for IP and western blot), MTA2 (Santa Cruz sc-28731), RBBP4 (Epitomics no. 2599-1), RBBP7 (Epitomics no. 3503-1), LSD1 (Abcam ab17721), BRG1 (Abcam ab4081 for immunostaining, a gift from Dr Wang for IP and western blot), KAP1 (Abcam ab22553), CREST (Antibodies Inc. no. 15-235), DIG (Roche no. 11093274910), and Flag M2 (Sigma F1804). .. Cells were fixed in 4% paraformaldehyde for 10 min at room temperature and permeabilized in 0.25% NP-40 in phosphate-buffered saline (PBS) for 10 min.

Transfection:

Article Title: Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells
Article Snippet: Briefly, 5637 cells were transfected with pll3.7-NC or pll3.7-iUCA1 viruses and selected with G418 for 5 days. .. ChIP was performed overnight at 4°C using the BRG1 antibody (ab4081; Abcam) or an isotype control IgG.

Article Title: BDNF rescues BAF53b-dependent synaptic plasticity and cocaine-associated memory in the nucleus accumbens
Article Snippet: Cells were harvested 48 h after transfection and lysed in TPER (ThermoFisher). .. Immunoprecipitations against Brg1 were performed using 1 μg of antibody ab70558 (Abcam,, Cambridge, MA).

Protease Inhibitor:

Article Title: RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells
Article Snippet: Cells were lysed with RIPA buffer (50 mM Tris-HCl pH 7.6, 1 % NP40, 0.5 % deoxycholic acid, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 % SDS) supplemented with Complete Protease Inhibitor Cocktail (Roche); lysate was incubated for 30 min on ice and sonicated three times for 10 sec each on medium power in order to reduce viscosity. .. The total protein extract (10 to 50 μg) was resolved on 8–10 % acrylamide gels, transferred on a nitrocellulose membrane (Hybond-c Extra, GE Healthcare), blocked with 5 % milk proteins in TBST (50 mM Tris pH 7.6, 150 mM NaCl, 0.05 % Tween-20), and probed with primary antibodies, including Dnmt3b (1:2000; Imgenex/Novus Biologicals; IMG-184A), SmarcA4 (1:1000; Abcam; ab4081), Kdm2b (1:2000; Merck Millipore; 09-864), PolII (clone CTD4H8; 1:1000; Millipore), α-tubulin (clone B-5-1-2; 1:5000; Sigma-Aldrich; T6074), GAPDH (Sigma-Aldrich; G9545), panAgo clone 2A8(1:250; Millipore; MABE56) and Ago2 clone 9E8.2 (1:2000; Millipore; 04-642).

Article Title: LXR-Mediated ABCA1 Expression and Function Are Modulated by High Glucose and PRMT2
Article Snippet: Macrophages were washed twice in ice-cold PBS prior to lysis (lysis buffer: 50 mM HEPES pH 7.6, 150 mM NaCl, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 1 mM NaF, 1% Triton X-100, 10% glycerol) with protease inhibitor cocktail (1:100, Cell Signaling). .. Antibodies used were anti-ABCA1 (1:1,000, Novus 400–105), anti-HSP90 (1:500, BD 610419), anti-LXRα (1:1,000, Abcam ab41902), anti-Myc (1:2,000, Cell Signaling 2276), and anti-dimethyl-arginine, asymmetric (ASYM25) (1:1,000, EMD Millipore 07–414), and anti-BRG1 (1:1,000, Abcam ab4081).

Cell Culture:

Article Title: The long non-coding RNA Dali is an epigenetic regulator of neural differentiation
Article Snippet: Paragraph title: Cell culture ... Biochemical fractionation, ChIP and UV-RIP experiments was performed exactly as described in The following antibodies were used: anti-DNMT1 (ab87656; Abcam, UK), anti-BRG1 (ab4081; Abcam), anti-P66beta (ab76924; Abcam), anti-SIN3A (Active Motif, Belgium, 39,865), anti-CTCF (Abcam, 70,303), anti-rabbit IgG control antibodies (Millipore, Billerica, MA) and mouse monoclonal anti-FLAG M2 beads (Sigma–Aldrich) for FLAG-tagged POU3F3 experiments.

Hemagglutination Assay:

Article Title: BRG1 promotes the repair of DNA double-strand breaks by facilitating the replacement of RPA with RAD51
Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-SMARCA4 (clone 20C3.2), anti-γH2AX (05-636) and rabbit polyclonal anti-H2A (07-146) (Millipore Corporation, CA); mouse monoclonal anti-HA (H9658), anti-GFP (G1546), anti-actin (A5441) and anti-BrdU (B2531) (Sigma-Aldrich, St Louis, MO); rabbit polyclonal anti-γH2AX (#9718S) (Cell Signaling Technology, Boston, MA); rabbit polyclonal anti-BRG1 (ab70558), anti-NBS1 (ab32074), anti-RAD51 (ab63801) and mouse monoclonal anti-RPA (ab2175) (Abcam, San Francisco, CA); rabbit polyclonal anti-H2AX (10856-1-AP) (Proteintech, Chicago, IL); rabbit polyclonal anti-RAD51 (sc-8349), anti-RAD52 (sc-365341), anti-BRCA2 (sc-28235) and mouse monoclonal anti-p53 (sc-126) (Santa Cruz Biotechnology, Dallas, TX). .. The following antibodies were used in this study: mouse monoclonal anti-SMARCA4 (clone 20C3.2), anti-γH2AX (05-636) and rabbit polyclonal anti-H2A (07-146) (Millipore Corporation, CA); mouse monoclonal anti-HA (H9658), anti-GFP (G1546), anti-actin (A5441) and anti-BrdU (B2531) (Sigma-Aldrich, St Louis, MO); rabbit polyclonal anti-γH2AX (#9718S) (Cell Signaling Technology, Boston, MA); rabbit polyclonal anti-BRG1 (ab70558), anti-NBS1 (ab32074), anti-RAD51 (ab63801) and mouse monoclonal anti-RPA (ab2175) (Abcam, San Francisco, CA); rabbit polyclonal anti-H2AX (10856-1-AP) (Proteintech, Chicago, IL); rabbit polyclonal anti-RAD51 (sc-8349), anti-RAD52 (sc-365341), anti-BRCA2 (sc-28235) and mouse monoclonal anti-p53 (sc-126) (Santa Cruz Biotechnology, Dallas, TX).

Light Microscopy:

Article Title: Bmi1 and BRG1 drive myocardial repair by regulating cardiac stem cell function in acute rheumatic heart disease
Article Snippet: Non-specific sites were blocked using 4% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) solution at room temperature for 1 h. Subsequently, the sections were treated with primary antibodies (1:400) directed against c-kit (cat. no. ab32363), Bmi1 (cat. no. ab38295) or BRG1 (cat. no. ab70558; all Abcam, Cambridge, UK) overnight at 4°C. .. Non-specific sites were blocked using 4% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) solution at room temperature for 1 h. Subsequently, the sections were treated with primary antibodies (1:400) directed against c-kit (cat. no. ab32363), Bmi1 (cat. no. ab38295) or BRG1 (cat. no. ab70558; all Abcam, Cambridge, UK) overnight at 4°C.

other:

Article Title: BRG1 Promotes chromatin remodeling around DNA damage sites
Article Snippet: An anti-BRG1 antibody (ab70558) was purchased from Abcam (San Francisco, CA).

Imaging:

Article Title: Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells
Article Snippet: Cells were lysed in RIPA buffer (Applygen, Beijing, China) and total cell lysates were separated by SDS-PAGE, transferred to PVDF membranes (Merck Millipore, Darmstadt, Germany), immunoblotted with antibodies, and visualized using a ChemiDoc XRS+ Imaging System (Bio-Rad) or film. .. Antibodies used for immunoblotting were anti-β-actin antibody (PM053; MBL, Japan) (1:5,000), anti-human p21 (3733-1; Abcam Epitomics, Cambridge, UK) (1:2,000), anti-H3K9me3 (49–1008; Novex, Carlsbad, CA, USA) (1:1,000), anti-H3K4m3 (ab8580) (1:2,000) and anti-human BRG1 antibodies (ab4081) (both from Abcam) (1:2,000).

Protein Concentration:

Article Title: SWI/SNF Mediates Polycomb Eviction and Epigenetic Reprogramming of the INK4b-ARF-INK4a Locus
Article Snippet: Cell extracts were prepared in radioimmunoprecipitation assay buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) and assayed for protein concentration, and 50 μg of extract was resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and then analyzed by immunoblotting. .. Primary antibodies included SNF5 (ab12167 [Abcam]), BRG1 (ab4081 [Abcam]), SUZ12 (ab12073 [Abcam]), BMI1 (ab14389 [Abcam]), EZH2 (Sc-25383 [Santa Cruz]), DNMT3B (IMG-184A [IMGENEX]), and histone H3 (ab1791 [Abcam]).

Polymerase Chain Reaction:

Article Title: Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells
Article Snippet: ChIP was performed overnight at 4°C using the BRG1 antibody (ab4081; Abcam) or an isotype control IgG. .. After a 1 h incubation in the presence of salmon sperm DNA/protein A agarose beads, the immunoprecipitated DNA/protein complexes were then washed and eluted from the beads with 1% SDS and 0.1 M NaHCO3 solution.

Sonication:

Article Title: RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells
Article Snippet: Cells were lysed with RIPA buffer (50 mM Tris-HCl pH 7.6, 1 % NP40, 0.5 % deoxycholic acid, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 % SDS) supplemented with Complete Protease Inhibitor Cocktail (Roche); lysate was incubated for 30 min on ice and sonicated three times for 10 sec each on medium power in order to reduce viscosity. .. The total protein extract (10 to 50 μg) was resolved on 8–10 % acrylamide gels, transferred on a nitrocellulose membrane (Hybond-c Extra, GE Healthcare), blocked with 5 % milk proteins in TBST (50 mM Tris pH 7.6, 150 mM NaCl, 0.05 % Tween-20), and probed with primary antibodies, including Dnmt3b (1:2000; Imgenex/Novus Biologicals; IMG-184A), SmarcA4 (1:1000; Abcam; ab4081), Kdm2b (1:2000; Merck Millipore; 09-864), PolII (clone CTD4H8; 1:1000; Millipore), α-tubulin (clone B-5-1-2; 1:5000; Sigma-Aldrich; T6074), GAPDH (Sigma-Aldrich; G9545), panAgo clone 2A8(1:250; Millipore; MABE56) and Ago2 clone 9E8.2 (1:2000; Millipore; 04-642).

Article Title: SWI/SNF Mediates Polycomb Eviction and Epigenetic Reprogramming of the INK4b-ARF-INK4a Locus
Article Snippet: Chromatin isolation, sonication yielding fragments of 300 to 600 bp, and immunoprecipitations were performed according to previously established protocols. .. The following antibodies were used: SNF5 (ab12167 [Abcam]), BRG1 (ab4081 [Abcam]), SUZ12 (ab12073 [Abcam]), BMI1 (ab14389 [Abcam]), EZH2 (Sc-25383 [Santa Cruz]), DNMT3B (IMG-184A [IMGENEX]), POL II (Sc-899 [Santa Cruz]), mixed lineage leukemia 1 (MLL1; A300-086A [Bethyl Laboratories]), Histone H3 (ab1791 [Abcam]), H3-K4me3 (ab12209 [Abcam]), and H3-K27me3 (catalog no. 07-449 [Upstate]).

Article Title: Involvement of Telomerase Reverse Transcriptase in Heterochromatin Maintenance
Article Snippet: The lysate was preabsorbed with 40 μl of Pierce protein A plus agarose (Thermo Scientific) for 30 min. Preabsorbed lysate was mixed with 10 μg of anti-hTERT MAb (clone 10E9-2), anti-BRG1 antibody (ab4081; Abcam), or anti-NS antibody (A300-599A; Bethyl Laboratories) and 40 μl of Pierce protein A plus agarose and incubated overnight at 4°C. .. The lysate was preabsorbed with 40 μl of Pierce protein A plus agarose (Thermo Scientific) for 30 min. Preabsorbed lysate was mixed with 10 μg of anti-hTERT MAb (clone 10E9-2), anti-BRG1 antibody (ab4081; Abcam), or anti-NS antibody (A300-599A; Bethyl Laboratories) and 40 μl of Pierce protein A plus agarose and incubated overnight at 4°C.

Methylated DNA Immunoprecipitation:

Article Title: SWI/SNF Mediates Polycomb Eviction and Epigenetic Reprogramming of the INK4b-ARF-INK4a Locus
Article Snippet: Paragraph title: ChIP and MeDIP assays. ... The following antibodies were used: SNF5 (ab12167 [Abcam]), BRG1 (ab4081 [Abcam]), SUZ12 (ab12073 [Abcam]), BMI1 (ab14389 [Abcam]), EZH2 (Sc-25383 [Santa Cruz]), DNMT3B (IMG-184A [IMGENEX]), POL II (Sc-899 [Santa Cruz]), mixed lineage leukemia 1 (MLL1; A300-086A [Bethyl Laboratories]), Histone H3 (ab1791 [Abcam]), H3-K4me3 (ab12209 [Abcam]), and H3-K27me3 (catalog no. 07-449 [Upstate]).

Nucleic Acid Electrophoresis:

Article Title: SWI/SNF Mediates Polycomb Eviction and Epigenetic Reprogramming of the INK4b-ARF-INK4a Locus
Article Snippet: Cell extracts were prepared in radioimmunoprecipitation assay buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) and assayed for protein concentration, and 50 μg of extract was resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and then analyzed by immunoblotting. .. Primary antibodies included SNF5 (ab12167 [Abcam]), BRG1 (ab4081 [Abcam]), SUZ12 (ab12073 [Abcam]), BMI1 (ab14389 [Abcam]), EZH2 (Sc-25383 [Santa Cruz]), DNMT3B (IMG-184A [IMGENEX]), and histone H3 (ab1791 [Abcam]).

Methylation:

Article Title: SWI/SNF Mediates Polycomb Eviction and Epigenetic Reprogramming of the INK4b-ARF-INK4a Locus
Article Snippet: The following antibodies were used: SNF5 (ab12167 [Abcam]), BRG1 (ab4081 [Abcam]), SUZ12 (ab12073 [Abcam]), BMI1 (ab14389 [Abcam]), EZH2 (Sc-25383 [Santa Cruz]), DNMT3B (IMG-184A [IMGENEX]), POL II (Sc-899 [Santa Cruz]), mixed lineage leukemia 1 (MLL1; A300-086A [Bethyl Laboratories]), Histone H3 (ab1791 [Abcam]), H3-K4me3 (ab12209 [Abcam]), and H3-K27me3 (catalog no. 07-449 [Upstate]). .. ChIP using species- and isotype-matched immunoglobulins directed against an unrelated protein (glutathione S -transferase [GST]) were used to determine background levels.

Isolation:

Article Title: SWI/SNF Mediates Polycomb Eviction and Epigenetic Reprogramming of the INK4b-ARF-INK4a Locus
Article Snippet: Chromatin isolation, sonication yielding fragments of 300 to 600 bp, and immunoprecipitations were performed according to previously established protocols. .. The following antibodies were used: SNF5 (ab12167 [Abcam]), BRG1 (ab4081 [Abcam]), SUZ12 (ab12073 [Abcam]), BMI1 (ab14389 [Abcam]), EZH2 (Sc-25383 [Santa Cruz]), DNMT3B (IMG-184A [IMGENEX]), POL II (Sc-899 [Santa Cruz]), mixed lineage leukemia 1 (MLL1; A300-086A [Bethyl Laboratories]), Histone H3 (ab1791 [Abcam]), H3-K4me3 (ab12209 [Abcam]), and H3-K27me3 (catalog no. 07-449 [Upstate]).

Chemiluminescence Immunoassay:

Article Title: LXR-Mediated ABCA1 Expression and Function Are Modulated by High Glucose and PRMT2
Article Snippet: Antibodies used were anti-ABCA1 (1:1,000, Novus 400–105), anti-HSP90 (1:500, BD 610419), anti-LXRα (1:1,000, Abcam ab41902), anti-Myc (1:2,000, Cell Signaling 2276), and anti-dimethyl-arginine, asymmetric (ASYM25) (1:1,000, EMD Millipore 07–414), and anti-BRG1 (1:1,000, Abcam ab4081). .. Membranes were incubated with horseradish peroxidase-conjugated secondary antibody in TBS-T at room temperature for 1 hour.

Immunohistochemistry:

Article Title: Bmi1 and BRG1 drive myocardial repair by regulating cardiac stem cell function in acute rheumatic heart disease
Article Snippet: Paragraph title: Histology and immunohistochemistry ... Non-specific sites were blocked using 4% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) solution at room temperature for 1 h. Subsequently, the sections were treated with primary antibodies (1:400) directed against c-kit (cat. no. ab32363), Bmi1 (cat. no. ab38295) or BRG1 (cat. no. ab70558; all Abcam, Cambridge, UK) overnight at 4°C.

Size-exclusion Chromatography:

Article Title: RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells
Article Snippet: Cells were lysed with RIPA buffer (50 mM Tris-HCl pH 7.6, 1 % NP40, 0.5 % deoxycholic acid, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 % SDS) supplemented with Complete Protease Inhibitor Cocktail (Roche); lysate was incubated for 30 min on ice and sonicated three times for 10 sec each on medium power in order to reduce viscosity. .. The total protein extract (10 to 50 μg) was resolved on 8–10 % acrylamide gels, transferred on a nitrocellulose membrane (Hybond-c Extra, GE Healthcare), blocked with 5 % milk proteins in TBST (50 mM Tris pH 7.6, 150 mM NaCl, 0.05 % Tween-20), and probed with primary antibodies, including Dnmt3b (1:2000; Imgenex/Novus Biologicals; IMG-184A), SmarcA4 (1:1000; Abcam; ab4081), Kdm2b (1:2000; Merck Millipore; 09-864), PolII (clone CTD4H8; 1:1000; Millipore), α-tubulin (clone B-5-1-2; 1:5000; Sigma-Aldrich; T6074), GAPDH (Sigma-Aldrich; G9545), panAgo clone 2A8(1:250; Millipore; MABE56) and Ago2 clone 9E8.2 (1:2000; Millipore; 04-642).

Article Title: Bmi1 and BRG1 drive myocardial repair by regulating cardiac stem cell function in acute rheumatic heart disease
Article Snippet: Non-specific sites were blocked using 4% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) solution at room temperature for 1 h. Subsequently, the sections were treated with primary antibodies (1:400) directed against c-kit (cat. no. ab32363), Bmi1 (cat. no. ab38295) or BRG1 (cat. no. ab70558; all Abcam, Cambridge, UK) overnight at 4°C. .. Non-specific sites were blocked using 4% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) solution at room temperature for 1 h. Subsequently, the sections were treated with primary antibodies (1:400) directed against c-kit (cat. no. ab32363), Bmi1 (cat. no. ab38295) or BRG1 (cat. no. ab70558; all Abcam, Cambridge, UK) overnight at 4°C.

Sequencing:

Article Title: MeCP2 interacts with chromosomal microRNAs in brain
Article Snippet: Paragraph title: RNA immunoprecipitation and massive parallel sequencing (RIP-seq) ... Antibodies used were anti-Brm (Abcam, ab15597), anti-Brg1 (Abcam, ab4081), anti-Dhx9 (ThermoFisher, PA5-19542), anti-Hp1 alpha (Abcam, ab77256), anti-MeCP2 antibody (M9317, Sigma), anti-Pol II (Millipore, 05-623).

Immunostaining:

Article Title: Transient bursts of Zscan4 expression are accompanied by the rapid derepression of heterochromatin in mouse embryonic stem cells
Article Snippet: The following antibodies were used: mouse polyclonal anti-Zscan4 (Abnova H00201516- B01P), rabbit polyclonal anti-Zscan4, HP1α (Millipore MAB3584 for immunostaining, Millipore no. 05-689 for immunoblotting), H3K9me3 (Upstate no. 07-442), H4K20me3 (Millipore no. 07-463), H3K4me3 (Millipore no. 07-473), H3K9ac (Millipore no. 07-352), H3K14ac (Millipore no. 17-10051), H3K18ac (Epitomics no. 1766-1), H3K27ac (Active Motif no. 39-133 for immunostaining and ChIP, Abcam ab4729 for immunostaining and immunoblotting), H3K9me2 (Abcam ab1220), pan-H3 (Abcam ab1791), p300 (Millipore no. 05-257), CBP (Santa Cruz sc-583), HDAC1 (Abcam ab7028 for immunostaining, ab31263 for western blot), HDAC2 (ab7029), MTA1 (Bethyl lab. .. A300-911A for immunostaining, Abcam ab71153 for IP and western blot), MTA2 (Santa Cruz sc-28731), RBBP4 (Epitomics no. 2599-1), RBBP7 (Epitomics no. 3503-1), LSD1 (Abcam ab17721), BRG1 (Abcam ab4081 for immunostaining, a gift from Dr Wang for IP and western blot), KAP1 (Abcam ab22553), CREST (Antibodies Inc. no. 15-235), DIG (Roche no. 11093274910), and Flag M2 (Sigma F1804). .. Cells were fixed in 4% paraformaldehyde for 10 min at room temperature and permeabilized in 0.25% NP-40 in phosphate-buffered saline (PBS) for 10 min.

Polyacrylamide Gel Electrophoresis:

Article Title: SWI/SNF Mediates Polycomb Eviction and Epigenetic Reprogramming of the INK4b-ARF-INK4a Locus
Article Snippet: Cell extracts were prepared in radioimmunoprecipitation assay buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) and assayed for protein concentration, and 50 μg of extract was resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and then analyzed by immunoblotting. .. Primary antibodies included SNF5 (ab12167 [Abcam]), BRG1 (ab4081 [Abcam]), SUZ12 (ab12073 [Abcam]), BMI1 (ab14389 [Abcam]), EZH2 (Sc-25383 [Santa Cruz]), DNMT3B (IMG-184A [IMGENEX]), and histone H3 (ab1791 [Abcam]).

Lysis:

Article Title: LXR-Mediated ABCA1 Expression and Function Are Modulated by High Glucose and PRMT2
Article Snippet: Macrophages were washed twice in ice-cold PBS prior to lysis (lysis buffer: 50 mM HEPES pH 7.6, 150 mM NaCl, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 1 mM NaF, 1% Triton X-100, 10% glycerol) with protease inhibitor cocktail (1:100, Cell Signaling). .. Antibodies used were anti-ABCA1 (1:1,000, Novus 400–105), anti-HSP90 (1:500, BD 610419), anti-LXRα (1:1,000, Abcam ab41902), anti-Myc (1:2,000, Cell Signaling 2276), and anti-dimethyl-arginine, asymmetric (ASYM25) (1:1,000, EMD Millipore 07–414), and anti-BRG1 (1:1,000, Abcam ab4081).

Article Title: Involvement of Telomerase Reverse Transcriptase in Heterochromatin Maintenance
Article Snippet: For the assay, 1 × 107 cells were lysed in 1 ml of lysis buffer A (0.5% NP-40, 20 mM Tris-HCl [pH 7.4], and 150 mM NaCl). .. The lysate was preabsorbed with 40 μl of Pierce protein A plus agarose (Thermo Scientific) for 30 min. Preabsorbed lysate was mixed with 10 μg of anti-hTERT MAb (clone 10E9-2), anti-BRG1 antibody (ab4081; Abcam), or anti-NS antibody (A300-599A; Bethyl Laboratories) and 40 μl of Pierce protein A plus agarose and incubated overnight at 4°C.

Purification:

Article Title: Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells
Article Snippet: ChIP was performed overnight at 4°C using the BRG1 antibody (ab4081; Abcam) or an isotype control IgG. .. After a 1 h incubation in the presence of salmon sperm DNA/protein A agarose beads, the immunoprecipitated DNA/protein complexes were then washed and eluted from the beads with 1% SDS and 0.1 M NaHCO3 solution.

Article Title: MeCP2 interacts with chromosomal microRNAs in brain
Article Snippet: Antibodies used were anti-Brm (Abcam, ab15597), anti-Brg1 (Abcam, ab4081), anti-Dhx9 (ThermoFisher, PA5-19542), anti-Hp1 alpha (Abcam, ab77256), anti-MeCP2 antibody (M9317, Sigma), anti-Pol II (Millipore, 05-623). .. Antibodies used were anti-Brm (Abcam, ab15597), anti-Brg1 (Abcam, ab4081), anti-Dhx9 (ThermoFisher, PA5-19542), anti-Hp1 alpha (Abcam, ab77256), anti-MeCP2 antibody (M9317, Sigma), anti-Pol II (Millipore, 05-623).

Chromatin Immunoprecipitation:

Article Title: Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells
Article Snippet: Chromatin DNA was sheared using ultrasound to a size of 0.5–1 kb. .. ChIP was performed overnight at 4°C using the BRG1 antibody (ab4081; Abcam) or an isotype control IgG. .. After a 1 h incubation in the presence of salmon sperm DNA/protein A agarose beads, the immunoprecipitated DNA/protein complexes were then washed and eluted from the beads with 1% SDS and 0.1 M NaHCO3 solution.

Article Title: DNA demethylation induces SALL4 gene re-expression in subgroups of hepatocellular carcinoma associated with Hepatitis B or C virus infection
Article Snippet: Paragraph title: Chromatin immunoprecipitation (ChIP) assays, immunoprecipitations and immunoblotting ... The following antibodies were used in this study: SALL4 antibody (Abcam, Cambridge, MA); HBc antibody (Dako, Carpinteria, CA); RNA polymerase II antibody (Santa cruz biotechnology, Dallas, Texas); BRG1 antibody (Abcam, Cambridge, MA); STAT3 antibody (Santa Cruz biotechnology, Dallas, Texas); OCT4 antibody (Abcam, Cambridge, MA); H3K27me3 antibody (Abcam, Cambridge, MA); Histone 3 antibody (Active Motif, Carlsbad, CA).

Article Title: SWI/SNF Mediates Polycomb Eviction and Epigenetic Reprogramming of the INK4b-ARF-INK4a Locus
Article Snippet: Paragraph title: ChIP and MeDIP assays. ... The following antibodies were used: SNF5 (ab12167 [Abcam]), BRG1 (ab4081 [Abcam]), SUZ12 (ab12073 [Abcam]), BMI1 (ab14389 [Abcam]), EZH2 (Sc-25383 [Santa Cruz]), DNMT3B (IMG-184A [IMGENEX]), POL II (Sc-899 [Santa Cruz]), mixed lineage leukemia 1 (MLL1; A300-086A [Bethyl Laboratories]), Histone H3 (ab1791 [Abcam]), H3-K4me3 (ab12209 [Abcam]), and H3-K27me3 (catalog no. 07-449 [Upstate]).

Article Title: The long non-coding RNA Dali is an epigenetic regulator of neural differentiation
Article Snippet: Human neuroblastoma (SH-SY5Y) cells were grown in DMEM/F12 medium supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% L-glutamine at 37°C in a humidified atmosphere with 5% CO2 . .. Biochemical fractionation, ChIP and UV-RIP experiments was performed exactly as described in The following antibodies were used: anti-DNMT1 (ab87656; Abcam, UK), anti-BRG1 (ab4081; Abcam), anti-P66beta (ab76924; Abcam), anti-SIN3A (Active Motif, Belgium, 39,865), anti-CTCF (Abcam, 70,303), anti-rabbit IgG control antibodies (Millipore, Billerica, MA) and mouse monoclonal anti-FLAG M2 beads (Sigma–Aldrich) for FLAG-tagged POU3F3 experiments. .. All animal experiments were conducted in accordance to schedule one UK Home Office guidelines (Scientific Procedures Act, 1986).

Article Title: MeCP2 interacts with chromosomal microRNAs in brain
Article Snippet: Antibodies used were anti-Brm (Abcam, ab15597), anti-Brg1 (Abcam, ab4081), anti-Dhx9 (ThermoFisher, PA5-19542), anti-Hp1 alpha (Abcam, ab77256), anti-MeCP2 antibody (M9317, Sigma), anti-Pol II (Millipore, 05-623). .. Chromatin Immunoprecipitation on MNase digested chromatin fractions.

Article Title: Transient bursts of Zscan4 expression are accompanied by the rapid derepression of heterochromatin in mouse embryonic stem cells
Article Snippet: The following antibodies were used: mouse polyclonal anti-Zscan4 (Abnova H00201516- B01P), rabbit polyclonal anti-Zscan4, HP1α (Millipore MAB3584 for immunostaining, Millipore no. 05-689 for immunoblotting), H3K9me3 (Upstate no. 07-442), H4K20me3 (Millipore no. 07-463), H3K4me3 (Millipore no. 07-473), H3K9ac (Millipore no. 07-352), H3K14ac (Millipore no. 17-10051), H3K18ac (Epitomics no. 1766-1), H3K27ac (Active Motif no. 39-133 for immunostaining and ChIP, Abcam ab4729 for immunostaining and immunoblotting), H3K9me2 (Abcam ab1220), pan-H3 (Abcam ab1791), p300 (Millipore no. 05-257), CBP (Santa Cruz sc-583), HDAC1 (Abcam ab7028 for immunostaining, ab31263 for western blot), HDAC2 (ab7029), MTA1 (Bethyl lab. .. A300-911A for immunostaining, Abcam ab71153 for IP and western blot), MTA2 (Santa Cruz sc-28731), RBBP4 (Epitomics no. 2599-1), RBBP7 (Epitomics no. 3503-1), LSD1 (Abcam ab17721), BRG1 (Abcam ab4081 for immunostaining, a gift from Dr Wang for IP and western blot), KAP1 (Abcam ab22553), CREST (Antibodies Inc. no. 15-235), DIG (Roche no. 11093274910), and Flag M2 (Sigma F1804).

SDS Page:

Article Title: Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells
Article Snippet: Cells were lysed in RIPA buffer (Applygen, Beijing, China) and total cell lysates were separated by SDS-PAGE, transferred to PVDF membranes (Merck Millipore, Darmstadt, Germany), immunoblotted with antibodies, and visualized using a ChemiDoc XRS+ Imaging System (Bio-Rad) or film. .. Antibodies used for immunoblotting were anti-β-actin antibody (PM053; MBL, Japan) (1:5,000), anti-human p21 (3733-1; Abcam Epitomics, Cambridge, UK) (1:2,000), anti-H3K9me3 (49–1008; Novex, Carlsbad, CA, USA) (1:1,000), anti-H3K4m3 (ab8580) (1:2,000) and anti-human BRG1 antibodies (ab4081) (both from Abcam) (1:2,000).

Article Title: The spring-loaded genome: Nucleosome redistributions are widespread, transient, and DNA-directed
Article Snippet: The BRG1 antibody (#4081) was purchased from Abcam. .. The BRG1 antibody (#4081) was purchased from Abcam.

Article Title: SNF5 Is an Essential Executor of Epigenetic Regulation during Differentiation
Article Snippet: Cell lysates were boiled in Laemmli sample buffer for 3 min, and 30 µg of each protein were subjected to SDS-PAGE. .. Antibodies against SNF5 (ab12167), BRG1 (ab4081), BRM (ab15597), and BAF53b (ab103771) were purchased from Abcam, Inc (Cambridge, MA).

Plasmid Preparation:

Article Title: BRG1 promotes the repair of DNA double-strand breaks by facilitating the replacement of RPA with RAD51
Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-SMARCA4 (clone 20C3.2), anti-γH2AX (05-636) and rabbit polyclonal anti-H2A (07-146) (Millipore Corporation, CA); mouse monoclonal anti-HA (H9658), anti-GFP (G1546), anti-actin (A5441) and anti-BrdU (B2531) (Sigma-Aldrich, St Louis, MO); rabbit polyclonal anti-γH2AX (#9718S) (Cell Signaling Technology, Boston, MA); rabbit polyclonal anti-BRG1 (ab70558), anti-NBS1 (ab32074), anti-RAD51 (ab63801) and mouse monoclonal anti-RPA (ab2175) (Abcam, San Francisco, CA); rabbit polyclonal anti-H2AX (10856-1-AP) (Proteintech, Chicago, IL); rabbit polyclonal anti-RAD51 (sc-8349), anti-RAD52 (sc-365341), anti-BRCA2 (sc-28235) and mouse monoclonal anti-p53 (sc-126) (Santa Cruz Biotechnology, Dallas, TX). .. The following antibodies were used in this study: mouse monoclonal anti-SMARCA4 (clone 20C3.2), anti-γH2AX (05-636) and rabbit polyclonal anti-H2A (07-146) (Millipore Corporation, CA); mouse monoclonal anti-HA (H9658), anti-GFP (G1546), anti-actin (A5441) and anti-BrdU (B2531) (Sigma-Aldrich, St Louis, MO); rabbit polyclonal anti-γH2AX (#9718S) (Cell Signaling Technology, Boston, MA); rabbit polyclonal anti-BRG1 (ab70558), anti-NBS1 (ab32074), anti-RAD51 (ab63801) and mouse monoclonal anti-RPA (ab2175) (Abcam, San Francisco, CA); rabbit polyclonal anti-H2AX (10856-1-AP) (Proteintech, Chicago, IL); rabbit polyclonal anti-RAD51 (sc-8349), anti-RAD52 (sc-365341), anti-BRCA2 (sc-28235) and mouse monoclonal anti-p53 (sc-126) (Santa Cruz Biotechnology, Dallas, TX).

Article Title: BDNF rescues BAF53b-dependent synaptic plasticity and cocaine-associated memory in the nucleus accumbens
Article Snippet: HT22 cells (Salk Institute, La Jolla, CA) were transfected with either the empty vector, pLVX-EF1a-IRES-mCherry, MW95 or MW97 using Lipofectamine LTX with Plus Reagent (ThermoFisher, Waltham, MA). .. Immunoprecipitations against Brg1 were performed using 1 μg of antibody ab70558 (Abcam,, Cambridge, MA).

Software:

Article Title: Bmi1 and BRG1 drive myocardial repair by regulating cardiac stem cell function in acute rheumatic heart disease
Article Snippet: Non-specific sites were blocked using 4% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) solution at room temperature for 1 h. Subsequently, the sections were treated with primary antibodies (1:400) directed against c-kit (cat. no. ab32363), Bmi1 (cat. no. ab38295) or BRG1 (cat. no. ab70558; all Abcam, Cambridge, UK) overnight at 4°C. .. Non-specific sites were blocked using 4% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) solution at room temperature for 1 h. Subsequently, the sections were treated with primary antibodies (1:400) directed against c-kit (cat. no. ab32363), Bmi1 (cat. no. ab38295) or BRG1 (cat. no. ab70558; all Abcam, Cambridge, UK) overnight at 4°C.

In Vitro:

Article Title: The long non-coding RNA Dali is an epigenetic regulator of neural differentiation
Article Snippet: The N2A cell line was chosen because it has been used extensively as a model to study neural differentiation in vitro ( ). .. Biochemical fractionation, ChIP and UV-RIP experiments was performed exactly as described in The following antibodies were used: anti-DNMT1 (ab87656; Abcam, UK), anti-BRG1 (ab4081; Abcam), anti-P66beta (ab76924; Abcam), anti-SIN3A (Active Motif, Belgium, 39,865), anti-CTCF (Abcam, 70,303), anti-rabbit IgG control antibodies (Millipore, Billerica, MA) and mouse monoclonal anti-FLAG M2 beads (Sigma–Aldrich) for FLAG-tagged POU3F3 experiments.

Radio Immunoprecipitation:

Article Title: SWI/SNF Mediates Polycomb Eviction and Epigenetic Reprogramming of the INK4b-ARF-INK4a Locus
Article Snippet: Cell extracts were prepared in radioimmunoprecipitation assay buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) and assayed for protein concentration, and 50 μg of extract was resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and then analyzed by immunoblotting. .. Primary antibodies included SNF5 (ab12167 [Abcam]), BRG1 (ab4081 [Abcam]), SUZ12 (ab12073 [Abcam]), BMI1 (ab14389 [Abcam]), EZH2 (Sc-25383 [Santa Cruz]), DNMT3B (IMG-184A [IMGENEX]), and histone H3 (ab1791 [Abcam]).

Ethanol Precipitation:

Article Title: SWI/SNF Mediates Polycomb Eviction and Epigenetic Reprogramming of the INK4b-ARF-INK4a Locus
Article Snippet: The following antibodies were used: SNF5 (ab12167 [Abcam]), BRG1 (ab4081 [Abcam]), SUZ12 (ab12073 [Abcam]), BMI1 (ab14389 [Abcam]), EZH2 (Sc-25383 [Santa Cruz]), DNMT3B (IMG-184A [IMGENEX]), POL II (Sc-899 [Santa Cruz]), mixed lineage leukemia 1 (MLL1; A300-086A [Bethyl Laboratories]), Histone H3 (ab1791 [Abcam]), H3-K4me3 (ab12209 [Abcam]), and H3-K27me3 (catalog no. 07-449 [Upstate]). .. ChIP using species- and isotype-matched immunoglobulins directed against an unrelated protein (glutathione S -transferase [GST]) were used to determine background levels.

Immunoprecipitation:

Article Title: BDNF rescues BAF53b-dependent synaptic plasticity and cocaine-associated memory in the nucleus accumbens
Article Snippet: Paragraph title: Immunoprecipitation and western blotting ... Immunoprecipitations against Brg1 were performed using 1 μg of antibody ab70558 (Abcam,, Cambridge, MA).

Article Title: MeCP2 interacts with chromosomal microRNAs in brain
Article Snippet: Paragraph title: RNA immunoprecipitation and massive parallel sequencing (RIP-seq) ... Antibodies used were anti-Brm (Abcam, ab15597), anti-Brg1 (Abcam, ab4081), anti-Dhx9 (ThermoFisher, PA5-19542), anti-Hp1 alpha (Abcam, ab77256), anti-MeCP2 antibody (M9317, Sigma), anti-Pol II (Millipore, 05-623).

Construct:

Article Title: BRG1 promotes the repair of DNA double-strand breaks by facilitating the replacement of RPA with RAD51
Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-SMARCA4 (clone 20C3.2), anti-γH2AX (05-636) and rabbit polyclonal anti-H2A (07-146) (Millipore Corporation, CA); mouse monoclonal anti-HA (H9658), anti-GFP (G1546), anti-actin (A5441) and anti-BrdU (B2531) (Sigma-Aldrich, St Louis, MO); rabbit polyclonal anti-γH2AX (#9718S) (Cell Signaling Technology, Boston, MA); rabbit polyclonal anti-BRG1 (ab70558), anti-NBS1 (ab32074), anti-RAD51 (ab63801) and mouse monoclonal anti-RPA (ab2175) (Abcam, San Francisco, CA); rabbit polyclonal anti-H2AX (10856-1-AP) (Proteintech, Chicago, IL); rabbit polyclonal anti-RAD51 (sc-8349), anti-RAD52 (sc-365341), anti-BRCA2 (sc-28235) and mouse monoclonal anti-p53 (sc-126) (Santa Cruz Biotechnology, Dallas, TX). .. The pBABE HA- Asi SI-ER plasmid was provided generously by Dr Gaelle Legube (Laboratoire de Biologie Cellulaire et Moleculaire du Controle de la Proliferation, France).

Fractionation:

Article Title: The long non-coding RNA Dali is an epigenetic regulator of neural differentiation
Article Snippet: Human neuroblastoma (SH-SY5Y) cells were grown in DMEM/F12 medium supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% L-glutamine at 37°C in a humidified atmosphere with 5% CO2 . .. Biochemical fractionation, ChIP and UV-RIP experiments was performed exactly as described in The following antibodies were used: anti-DNMT1 (ab87656; Abcam, UK), anti-BRG1 (ab4081; Abcam), anti-P66beta (ab76924; Abcam), anti-SIN3A (Active Motif, Belgium, 39,865), anti-CTCF (Abcam, 70,303), anti-rabbit IgG control antibodies (Millipore, Billerica, MA) and mouse monoclonal anti-FLAG M2 beads (Sigma–Aldrich) for FLAG-tagged POU3F3 experiments. .. All animal experiments were conducted in accordance to schedule one UK Home Office guidelines (Scientific Procedures Act, 1986).

Staining:

Article Title: The dilemma of early preventive oophorectomy in familial small cell carcinoma of the ovary of hypercalcemic type
Article Snippet: The expression of SMARCA4 (BRG1) protein was assessed immunohistochemically. .. Four-μm sections were cut from both ovaries and slides were stained with BRG1 antibody (Abcam; pretreatment ER2 20 min; dilution 1:100). .. Standard immunohistochemical methods were employed, including appropriate positive and negative tissue controls.

Article Title: Bmi1 and BRG1 drive myocardial repair by regulating cardiac stem cell function in acute rheumatic heart disease
Article Snippet: Non-specific sites were blocked using 4% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) solution at room temperature for 1 h. Subsequently, the sections were treated with primary antibodies (1:400) directed against c-kit (cat. no. ab32363), Bmi1 (cat. no. ab38295) or BRG1 (cat. no. ab70558; all Abcam, Cambridge, UK) overnight at 4°C. .. Non-specific sites were blocked using 4% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) solution at room temperature for 1 h. Subsequently, the sections were treated with primary antibodies (1:400) directed against c-kit (cat. no. ab32363), Bmi1 (cat. no. ab38295) or BRG1 (cat. no. ab70558; all Abcam, Cambridge, UK) overnight at 4°C.

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    Abcam anti brg1 antibody
    Summary model. (A) Dynamic expression of <t>Brg1</t> during cardiac differentiation peaks at the mesoderm stage. Brg1 function is most crucial during the mesoderm-induction stage. (B,C) Mechanisms of Brg1 function during mesoderm induction. (B) Brg1 is required for silent or poised enhancer to transition to an H3K27ac + active state. (C) Brg1 is required at non-mesodermal genes to promote Polycomb complex-mediated repression.
    Anti Brg1 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 85/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Summary model. (A) Dynamic expression of Brg1 during cardiac differentiation peaks at the mesoderm stage. Brg1 function is most crucial during the mesoderm-induction stage. (B,C) Mechanisms of Brg1 function during mesoderm induction. (B) Brg1 is required for silent or poised enhancer to transition to an H3K27ac + active state. (C) Brg1 is required at non-mesodermal genes to promote Polycomb complex-mediated repression.

    Journal: Development (Cambridge, England)

    Article Title: Brg1 modulates enhancer activation in mesoderm lineage commitment

    doi: 10.1242/dev.109496

    Figure Lengend Snippet: Summary model. (A) Dynamic expression of Brg1 during cardiac differentiation peaks at the mesoderm stage. Brg1 function is most crucial during the mesoderm-induction stage. (B,C) Mechanisms of Brg1 function during mesoderm induction. (B) Brg1 is required for silent or poised enhancer to transition to an H3K27ac + active state. (C) Brg1 is required at non-mesodermal genes to promote Polycomb complex-mediated repression.

    Article Snippet: Brg1 ChIP-exo was performed as previously described ( ) using anti-Brg1 antibody (Abcam, 110641; 3 µg).

    Techniques: Expressing

    Brg1 is required for robust induction of mesodermal markers. (A) Flow cytometry for Pdgfra and Flk-1 at day 4 of differentiation demonstrates a lower percentage of cells expressing these mesoderm markers in cultures deleted for Brg1 . (B) Quantitative PCR demonstrates reduced expression of Mesp1 in day 4 cultures depleted for Brg1 . *** P

    Journal: Development (Cambridge, England)

    Article Title: Brg1 modulates enhancer activation in mesoderm lineage commitment

    doi: 10.1242/dev.109496

    Figure Lengend Snippet: Brg1 is required for robust induction of mesodermal markers. (A) Flow cytometry for Pdgfra and Flk-1 at day 4 of differentiation demonstrates a lower percentage of cells expressing these mesoderm markers in cultures deleted for Brg1 . (B) Quantitative PCR demonstrates reduced expression of Mesp1 in day 4 cultures depleted for Brg1 . *** P

    Article Snippet: Brg1 ChIP-exo was performed as previously described ( ) using anti-Brg1 antibody (Abcam, 110641; 3 µg).

    Techniques: Flow Cytometry, Cytometry, Expressing, Real-time Polymerase Chain Reaction

    Brg1 is required for H3K27me3 levels at derepressed developmental regulators. (A) Density of ChIP-seq reads for H3K27ac and H3K27me3±5 kb of the TSS of upregulated genes. Regions are ranked according to H3K27me3 density. Right bar indicates distribution of developmental transcription factors (TFs). Most developmental TFs are marked by H3K27me3 and not by H3K27ac. (B) Example genomic regions with loss of H3K27me3 at derepressed genes. y -axis denotes reads per bin per million. (C,D) (Left) Average ChIP-seq or input signal from control or 4-OHT-treated cultures at promoters of group I genes for (C) H3K27me3 or (D) Suz12. (Right) Box plot of normalized (C) H3K27me3 or (D) Suz12 ChIPseq read density at group I gene promoters (±5 kb of TSS) for day 4 control (blue) or day 4 4-OHT (red). Significance between groups was determined using a two-sided paired Mann–Whitney U -test.

    Journal: Development (Cambridge, England)

    Article Title: Brg1 modulates enhancer activation in mesoderm lineage commitment

    doi: 10.1242/dev.109496

    Figure Lengend Snippet: Brg1 is required for H3K27me3 levels at derepressed developmental regulators. (A) Density of ChIP-seq reads for H3K27ac and H3K27me3±5 kb of the TSS of upregulated genes. Regions are ranked according to H3K27me3 density. Right bar indicates distribution of developmental transcription factors (TFs). Most developmental TFs are marked by H3K27me3 and not by H3K27ac. (B) Example genomic regions with loss of H3K27me3 at derepressed genes. y -axis denotes reads per bin per million. (C,D) (Left) Average ChIP-seq or input signal from control or 4-OHT-treated cultures at promoters of group I genes for (C) H3K27me3 or (D) Suz12. (Right) Box plot of normalized (C) H3K27me3 or (D) Suz12 ChIPseq read density at group I gene promoters (±5 kb of TSS) for day 4 control (blue) or day 4 4-OHT (red). Significance between groups was determined using a two-sided paired Mann–Whitney U -test.

    Article Snippet: Brg1 ChIP-exo was performed as previously described ( ) using anti-Brg1 antibody (Abcam, 110641; 3 µg).

    Techniques: Chromatin Immunoprecipitation, MANN-WHITNEY

    Brg1 co-localizes with H3K27ac genome-wide. (A) Density of ChIP-seq reads for H3K27ac, anti-FLAG ChIP-seq and anti-Brg1 ChIP-exo in Brg1-FLAG ESCs, and anti-Brg1 ChIP-exo in THF- and 4-OHT-treated Brg1 fl/fl ESCs. Plots show ±5 kb around the midpoint of each Brg1-enriched region ranked according to H3K27ac density, and demonstrate significant co-localization of Brg1 with H3K27ac. Brg1 is detected at these regions across distinct cell lines and ChIP antibodies, and is lost upon genetic deletion. (B) Distribution of Brg1-enriched regions across the mouse genome. (C) Overlap between genes with Brg1 enrichment within 2.5 kb of the TSS and genes dysregulated by loss of Brg1 demonstrates little overlap between Brg1 -dependent genes and Brg1-bound promoters. (D) Co-localization of Brg1 and H3K27ac at example genomic regions. y -axis shows reads per bin per million. (E) Motifs enriched at Brg1-occupied enhancer elements. TRANSFAC positional Weight Matrices for each significantly (q

    Journal: Development (Cambridge, England)

    Article Title: Brg1 modulates enhancer activation in mesoderm lineage commitment

    doi: 10.1242/dev.109496

    Figure Lengend Snippet: Brg1 co-localizes with H3K27ac genome-wide. (A) Density of ChIP-seq reads for H3K27ac, anti-FLAG ChIP-seq and anti-Brg1 ChIP-exo in Brg1-FLAG ESCs, and anti-Brg1 ChIP-exo in THF- and 4-OHT-treated Brg1 fl/fl ESCs. Plots show ±5 kb around the midpoint of each Brg1-enriched region ranked according to H3K27ac density, and demonstrate significant co-localization of Brg1 with H3K27ac. Brg1 is detected at these regions across distinct cell lines and ChIP antibodies, and is lost upon genetic deletion. (B) Distribution of Brg1-enriched regions across the mouse genome. (C) Overlap between genes with Brg1 enrichment within 2.5 kb of the TSS and genes dysregulated by loss of Brg1 demonstrates little overlap between Brg1 -dependent genes and Brg1-bound promoters. (D) Co-localization of Brg1 and H3K27ac at example genomic regions. y -axis shows reads per bin per million. (E) Motifs enriched at Brg1-occupied enhancer elements. TRANSFAC positional Weight Matrices for each significantly (q

    Article Snippet: Brg1 ChIP-exo was performed as previously described ( ) using anti-Brg1 antibody (Abcam, 110641; 3 µg).

    Techniques: Genome Wide, Chromatin Immunoprecipitation

    Brg1 is required for enhancer activation in differentiating mesodermal cultures. (A) Histogram of log 2 -fold change in H3K27ac at predicted enhancers. (B) Scatterplot of log 2 -fold change of H3K27ac at predicted enhancers and the log 2 -fold change in gene expression between day 4 4-OHT and day 4 control cultures of the nearest gene to each enhancer plotted. Red and blue dots highlight enhancers marked by H3K27ac in undifferentiated ESCs and mesodermal cultures (static enhancers), and those marked in mesoderm cultures only (activated enhancers), respectively. (C) Box plots of log 2 -fold change of subsets of predicted enhancers with read density profiles of each enhancer cohort. Enhancers associated with downregulated genes include enhancers of which the most proximal gene is significantly downregulated in Brg1 -deleted mesoderm. Downregulated gene-associated enhancers show greater average loss in H3K27ac than all enhancers. (D) Box plots of log 2 -fold change in H3K27ac for predicted enhancers in Brg1 -deficient cultures. Enhancers are separated into Brg1-bound and unbound cohorts based on the presence or absence of a Brg1-enriched region, respectively. (E,F) Box plots of log 2 -fold change in H3K27ac (E) or expression of the nearest gene (F) for static and activated enhancers in Brg1 -deficient cultures. (G) H3K27ac at putative enhancer regions proximal to the Mesp1 and Cyp26a1 genes. y -axis shows reads per bin per million.

    Journal: Development (Cambridge, England)

    Article Title: Brg1 modulates enhancer activation in mesoderm lineage commitment

    doi: 10.1242/dev.109496

    Figure Lengend Snippet: Brg1 is required for enhancer activation in differentiating mesodermal cultures. (A) Histogram of log 2 -fold change in H3K27ac at predicted enhancers. (B) Scatterplot of log 2 -fold change of H3K27ac at predicted enhancers and the log 2 -fold change in gene expression between day 4 4-OHT and day 4 control cultures of the nearest gene to each enhancer plotted. Red and blue dots highlight enhancers marked by H3K27ac in undifferentiated ESCs and mesodermal cultures (static enhancers), and those marked in mesoderm cultures only (activated enhancers), respectively. (C) Box plots of log 2 -fold change of subsets of predicted enhancers with read density profiles of each enhancer cohort. Enhancers associated with downregulated genes include enhancers of which the most proximal gene is significantly downregulated in Brg1 -deleted mesoderm. Downregulated gene-associated enhancers show greater average loss in H3K27ac than all enhancers. (D) Box plots of log 2 -fold change in H3K27ac for predicted enhancers in Brg1 -deficient cultures. Enhancers are separated into Brg1-bound and unbound cohorts based on the presence or absence of a Brg1-enriched region, respectively. (E,F) Box plots of log 2 -fold change in H3K27ac (E) or expression of the nearest gene (F) for static and activated enhancers in Brg1 -deficient cultures. (G) H3K27ac at putative enhancer regions proximal to the Mesp1 and Cyp26a1 genes. y -axis shows reads per bin per million.

    Article Snippet: Brg1 ChIP-exo was performed as previously described ( ) using anti-Brg1 antibody (Abcam, 110641; 3 µg).

    Techniques: Activation Assay, Expressing

    Global expression analysis of Brg1 -deleted mesodermal cultures reveals dysregulation of essential developmental genes. (A) Cartoon of the RNA-seq experimental design. (B) Day 4 expression in control samples plotted versus day 4 expression in 4-OHT-treated samples. Genes significantly changed ( > twofold change, FDR=1%) are colored in red and green for upregulated and downregulated, respectively. Example genes are highlighted.

    Journal: Development (Cambridge, England)

    Article Title: Brg1 modulates enhancer activation in mesoderm lineage commitment

    doi: 10.1242/dev.109496

    Figure Lengend Snippet: Global expression analysis of Brg1 -deleted mesodermal cultures reveals dysregulation of essential developmental genes. (A) Cartoon of the RNA-seq experimental design. (B) Day 4 expression in control samples plotted versus day 4 expression in 4-OHT-treated samples. Genes significantly changed ( > twofold change, FDR=1%) are colored in red and green for upregulated and downregulated, respectively. Example genes are highlighted.

    Article Snippet: Brg1 ChIP-exo was performed as previously described ( ) using anti-Brg1 antibody (Abcam, 110641; 3 µg).

    Techniques: Expressing, RNA Sequencing Assay

    Brg1 is required for gene activation and maintenance of gene silencing during mesoderm differentiation. (A) Heat map of log 2 -fold change in expression during mesoderm differentiation for genes significantly downregulated or upregulated by loss of Brg1 . (B) (Top) Genes are rank-ordered based on log 2 -fold change in expression between day 4 control and day 2 (normal mesoderm differentiation). Only genes significantly changed during mesoderm differentiation are shown. (Bottom) Color bars depict distribution of genes downregulated by loss of Brg1 by numerous fold change cut-offs. Colors vary only for ease of visualization and do not correlate to numerical values. Each vertical line represents a single dysregulated gene. Genes downregulated by loss of Brg1 cluster to the right, suggesting a role for Brg1 in gene activation during mesoderm differentiation. Conversely, genes upregulated by loss of Brg1 are more often found repressed during mesoderm differentiation. N indicates the number of Brg1 -dependent genes shown.

    Journal: Development (Cambridge, England)

    Article Title: Brg1 modulates enhancer activation in mesoderm lineage commitment

    doi: 10.1242/dev.109496

    Figure Lengend Snippet: Brg1 is required for gene activation and maintenance of gene silencing during mesoderm differentiation. (A) Heat map of log 2 -fold change in expression during mesoderm differentiation for genes significantly downregulated or upregulated by loss of Brg1 . (B) (Top) Genes are rank-ordered based on log 2 -fold change in expression between day 4 control and day 2 (normal mesoderm differentiation). Only genes significantly changed during mesoderm differentiation are shown. (Bottom) Color bars depict distribution of genes downregulated by loss of Brg1 by numerous fold change cut-offs. Colors vary only for ease of visualization and do not correlate to numerical values. Each vertical line represents a single dysregulated gene. Genes downregulated by loss of Brg1 cluster to the right, suggesting a role for Brg1 in gene activation during mesoderm differentiation. Conversely, genes upregulated by loss of Brg1 are more often found repressed during mesoderm differentiation. N indicates the number of Brg1 -dependent genes shown.

    Article Snippet: Brg1 ChIP-exo was performed as previously described ( ) using anti-Brg1 antibody (Abcam, 110641; 3 µg).

    Techniques: Activation Assay, Expressing

    Brg1 is required for directed differentiation of ESCs to cardiomyocytes (CMs). (A) Heat map representation of clustering analysis of RNA expression of chromatin regulators at four stages of directed CM differentiation identifies three expression patterns. Chromatin regulators include genes annotated with GO terms GO0006338 and GO00016569 and additional known regulators. (B) Western blot analysis demonstrates reduced abundance of Brg1 at late stages of CM differentiation. Lysate from the adrenal carcinoma SW13, which does not express Brg1, was used as a negative control. Actin was used as a loading control. (C) Western blot analysis of a 4-OHT treatment time course in Brg1 f/f ; Actin-CreER ESCs. Loss of Brg1 expression is near complete after 48 h of 4-OHT treatment. (D) Control (vehicle only, THF) or 4-OHT was added after 2, 4 and 8 days of differentiation to mediate Brg1 deletion, and the presence of cardiomyocytes was determined by immunofluorescence for cTnT at day 12. Scale bar: 50 µm. (E) Comparison of percentage of cTnT + cells for control or 4-OHT-treated cultures measured by flow cytometry. * P

    Journal: Development (Cambridge, England)

    Article Title: Brg1 modulates enhancer activation in mesoderm lineage commitment

    doi: 10.1242/dev.109496

    Figure Lengend Snippet: Brg1 is required for directed differentiation of ESCs to cardiomyocytes (CMs). (A) Heat map representation of clustering analysis of RNA expression of chromatin regulators at four stages of directed CM differentiation identifies three expression patterns. Chromatin regulators include genes annotated with GO terms GO0006338 and GO00016569 and additional known regulators. (B) Western blot analysis demonstrates reduced abundance of Brg1 at late stages of CM differentiation. Lysate from the adrenal carcinoma SW13, which does not express Brg1, was used as a negative control. Actin was used as a loading control. (C) Western blot analysis of a 4-OHT treatment time course in Brg1 f/f ; Actin-CreER ESCs. Loss of Brg1 expression is near complete after 48 h of 4-OHT treatment. (D) Control (vehicle only, THF) or 4-OHT was added after 2, 4 and 8 days of differentiation to mediate Brg1 deletion, and the presence of cardiomyocytes was determined by immunofluorescence for cTnT at day 12. Scale bar: 50 µm. (E) Comparison of percentage of cTnT + cells for control or 4-OHT-treated cultures measured by flow cytometry. * P

    Article Snippet: Brg1 ChIP-exo was performed as previously described ( ) using anti-Brg1 antibody (Abcam, 110641; 3 µg).

    Techniques: RNA Expression, Expressing, Western Blot, Negative Control, Immunofluorescence, Flow Cytometry, Cytometry

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-019-08380-1

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

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

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

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-019-08380-1

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

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

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

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-019-08380-1

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

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

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

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-019-08380-1

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

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

    Techniques: In Vivo

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-019-08380-1

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

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

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

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-019-08380-1

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

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

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

    Brg1 KO arrested meiosis at pachytene stage IV. A – B ) Lack of diplotene spermatocytes in Brg1 KO mice. Spermatocytes were stained with anti-synaptonemal complex protein 3 (SCP3) before the cells at the various stages of meiosis were enumerated

    Journal:

    Article Title: Essential Roles of the Chromatin Remodeling Factor Brg1 in Spermatogenesis in Mice

    doi: 10.1095/biolreprod.111.097097

    Figure Lengend Snippet: Brg1 KO arrested meiosis at pachytene stage IV. A – B ) Lack of diplotene spermatocytes in Brg1 KO mice. Spermatocytes were stained with anti-synaptonemal complex protein 3 (SCP3) before the cells at the various stages of meiosis were enumerated

    Article Snippet: Primary antibodies used were anti-EE2 (1:100; gift of Dr. Hiromitsu Tanaka), anti-BRG1 (1:100; 110641; Abcam), anti-BRM (1:100; 15597; Abcam), anti-H3K9me3 (1:100; 8898; Abcam), anti-phospho-S5 Pol II (1:100; IHC-00387; Bethyl Laboratories), anti-cH2AX (1:100; 05–636; Millipore), anti-RAD51 (1:100; 63801; Abcam), and anti-H1t (1:400; gift of Dr. Mary Ann Handel).

    Techniques: Gene Knockout, Mouse Assay, Staining

    Reduction of EE2+ cells in Brg1 KO testis. Testis sections were stained with anti-EE2 and counterstained with DAPI. EE2+ cells were visualized with immunofluorescence and counted, and the results displayed in the bar graph at the bottom. Six seminiferous

    Journal:

    Article Title: Essential Roles of the Chromatin Remodeling Factor Brg1 in Spermatogenesis in Mice

    doi: 10.1095/biolreprod.111.097097

    Figure Lengend Snippet: Reduction of EE2+ cells in Brg1 KO testis. Testis sections were stained with anti-EE2 and counterstained with DAPI. EE2+ cells were visualized with immunofluorescence and counted, and the results displayed in the bar graph at the bottom. Six seminiferous

    Article Snippet: Primary antibodies used were anti-EE2 (1:100; gift of Dr. Hiromitsu Tanaka), anti-BRG1 (1:100; 110641; Abcam), anti-BRM (1:100; 15597; Abcam), anti-H3K9me3 (1:100; 8898; Abcam), anti-phospho-S5 Pol II (1:100; IHC-00387; Bethyl Laboratories), anti-cH2AX (1:100; 05–636; Millipore), anti-RAD51 (1:100; 63801; Abcam), and anti-H1t (1:400; gift of Dr. Mary Ann Handel).

    Techniques: Gene Knockout, Staining, Immunofluorescence

    Potential defects in DSB repair in Brg1 KO testes. Testicular sections were stained for γH2AX (left two columns: a , b , e , f ) and RAD51 (right two columns: c , d , g , h ). The white and red arrows indicate examples of tubular sections containing pachytene

    Journal:

    Article Title: Essential Roles of the Chromatin Remodeling Factor Brg1 in Spermatogenesis in Mice

    doi: 10.1095/biolreprod.111.097097

    Figure Lengend Snippet: Potential defects in DSB repair in Brg1 KO testes. Testicular sections were stained for γH2AX (left two columns: a , b , e , f ) and RAD51 (right two columns: c , d , g , h ). The white and red arrows indicate examples of tubular sections containing pachytene

    Article Snippet: Primary antibodies used were anti-EE2 (1:100; gift of Dr. Hiromitsu Tanaka), anti-BRG1 (1:100; 110641; Abcam), anti-BRM (1:100; 15597; Abcam), anti-H3K9me3 (1:100; 8898; Abcam), anti-phospho-S5 Pol II (1:100; IHC-00387; Bethyl Laboratories), anti-cH2AX (1:100; 05–636; Millipore), anti-RAD51 (1:100; 63801; Abcam), and anti-H1t (1:400; gift of Dr. Mary Ann Handel).

    Techniques: Gene Knockout, Staining

    BRG1 and BRM expression in the testis. A ) BRG1 expression during spermatogenesis. Shown are BRG1 immunofluorescence (left), DAPI-stained nuclei (middle), and overlay of the two images (right). B ) BRG1 expression in primary spermatocytes. Cells were stained

    Journal:

    Article Title: Essential Roles of the Chromatin Remodeling Factor Brg1 in Spermatogenesis in Mice

    doi: 10.1095/biolreprod.111.097097

    Figure Lengend Snippet: BRG1 and BRM expression in the testis. A ) BRG1 expression during spermatogenesis. Shown are BRG1 immunofluorescence (left), DAPI-stained nuclei (middle), and overlay of the two images (right). B ) BRG1 expression in primary spermatocytes. Cells were stained

    Article Snippet: Primary antibodies used were anti-EE2 (1:100; gift of Dr. Hiromitsu Tanaka), anti-BRG1 (1:100; 110641; Abcam), anti-BRM (1:100; 15597; Abcam), anti-H3K9me3 (1:100; 8898; Abcam), anti-phospho-S5 Pol II (1:100; IHC-00387; Bethyl Laboratories), anti-cH2AX (1:100; 05–636; Millipore), anti-RAD51 (1:100; 63801; Abcam), and anti-H1t (1:400; gift of Dr. Mary Ann Handel).

    Techniques: Expressing, Immunofluorescence, Staining

    Sex body defects in Brg1 KO testes. A ) Testicular sections were stained for γH2AX and Pol II (S5), the Ser-5 phosphorylated form of RNA Pol II. B ) Same as A except that H3K9m3 instead of Pol II (S5) was analyzed. The insets are close-up views

    Journal:

    Article Title: Essential Roles of the Chromatin Remodeling Factor Brg1 in Spermatogenesis in Mice

    doi: 10.1095/biolreprod.111.097097

    Figure Lengend Snippet: Sex body defects in Brg1 KO testes. A ) Testicular sections were stained for γH2AX and Pol II (S5), the Ser-5 phosphorylated form of RNA Pol II. B ) Same as A except that H3K9m3 instead of Pol II (S5) was analyzed. The insets are close-up views

    Article Snippet: Primary antibodies used were anti-EE2 (1:100; gift of Dr. Hiromitsu Tanaka), anti-BRG1 (1:100; 110641; Abcam), anti-BRM (1:100; 15597; Abcam), anti-H3K9me3 (1:100; 8898; Abcam), anti-phospho-S5 Pol II (1:100; IHC-00387; Bethyl Laboratories), anti-cH2AX (1:100; 05–636; Millipore), anti-RAD51 (1:100; 63801; Abcam), and anti-H1t (1:400; gift of Dr. Mary Ann Handel).

    Techniques: Gene Knockout, Staining

    Spermatocyte apoptosis in Brg1 KO testis. A ) Apoptotic cells were visualized using TUNEL assays detecting DNA breaks. B ) FACS analysis of apoptosis based on DNA content and cell morphology. Testis single cell suspension was stained with PI to reveal haploid

    Journal:

    Article Title: Essential Roles of the Chromatin Remodeling Factor Brg1 in Spermatogenesis in Mice

    doi: 10.1095/biolreprod.111.097097

    Figure Lengend Snippet: Spermatocyte apoptosis in Brg1 KO testis. A ) Apoptotic cells were visualized using TUNEL assays detecting DNA breaks. B ) FACS analysis of apoptosis based on DNA content and cell morphology. Testis single cell suspension was stained with PI to reveal haploid

    Article Snippet: Primary antibodies used were anti-EE2 (1:100; gift of Dr. Hiromitsu Tanaka), anti-BRG1 (1:100; 110641; Abcam), anti-BRM (1:100; 15597; Abcam), anti-H3K9me3 (1:100; 8898; Abcam), anti-phospho-S5 Pol II (1:100; IHC-00387; Bethyl Laboratories), anti-cH2AX (1:100; 05–636; Millipore), anti-RAD51 (1:100; 63801; Abcam), and anti-H1t (1:400; gift of Dr. Mary Ann Handel).

    Techniques: Gene Knockout, TUNEL Assay, FACS, Staining

    Brg1 is essential for the development of male germline. Shown are gross pictures (top) and H E stained histological sections (bottom) of WT and Brg1 KO testes from 5-mo-old mice. Bar = 25 μm.

    Journal:

    Article Title: Essential Roles of the Chromatin Remodeling Factor Brg1 in Spermatogenesis in Mice

    doi: 10.1095/biolreprod.111.097097

    Figure Lengend Snippet: Brg1 is essential for the development of male germline. Shown are gross pictures (top) and H E stained histological sections (bottom) of WT and Brg1 KO testes from 5-mo-old mice. Bar = 25 μm.

    Article Snippet: Primary antibodies used were anti-EE2 (1:100; gift of Dr. Hiromitsu Tanaka), anti-BRG1 (1:100; 110641; Abcam), anti-BRM (1:100; 15597; Abcam), anti-H3K9me3 (1:100; 8898; Abcam), anti-phospho-S5 Pol II (1:100; IHC-00387; Bethyl Laboratories), anti-cH2AX (1:100; 05–636; Millipore), anti-RAD51 (1:100; 63801; Abcam), and anti-H1t (1:400; gift of Dr. Mary Ann Handel).

    Techniques: Staining, Gene Knockout, Mouse Assay

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

    Journal: Cell Death & Disease

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

    doi: 10.1038/cddis.2017.236

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

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

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

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

    Journal: Cell Death & Disease

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

    doi: 10.1038/cddis.2017.236

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

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

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

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

    Journal: Cell Death & Disease

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

    doi: 10.1038/cddis.2017.236

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

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

    Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction

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

    Journal: Cell Death & Disease

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

    doi: 10.1038/cddis.2017.236

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

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

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

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

    Journal: Cell Death & Disease

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

    doi: 10.1038/cddis.2017.236

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

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

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

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

    Journal: Cell Death & Disease

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

    doi: 10.1038/cddis.2017.236

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

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

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