Journal: Redox Biology

Article Title: Brd4 inhibition attenuates unilateral ureteral obstruction-induced fibrosis by blocking TGF-β-mediated Nox4 expression

doi: 10.1016/j.redox.2016.12.031

Figure Lengend Snippet: Brd4 was up-regulated in the kidney after unilateral ureteral obstruction (UUO). (A) Brd4 mRNA levels were detected by real-time RT-PCR at day 1 and 7 after UUO. (B) Brd4 protein levels were detected by western blot analysis at day 1 and 7 after UUO. (C) Immunohistochemical staining of Brd4 in renal sections at day 1 and 7 after UUO. Values are expressed as the mean±SEM. * P <0.05, relative to the sham group, n=3.

Article Snippet: The rabbit anti-Brd4 antibody was supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Quantitative RT-PCR, Western Blot, Immunohistochemical staining, Staining

Journal: Redox Biology

Article Title: Brd4 inhibition attenuates unilateral ureteral obstruction-induced fibrosis by blocking TGF-β-mediated Nox4 expression

doi: 10.1016/j.redox.2016.12.031

Figure Lengend Snippet: Brd4 inhibition attenuated TGF-β1-induced fibrosis and oxidative stress in HK-2 cells. (A-C) HK-2 cells were pretreated with or without JQ1 at different doses (0.1, 0.2, 0.5 μM) for 1 h, and then treated with TGF-β1 (10 ng/mL) for 24 h. (A) Real-time PCR analyses for the mRNA expression of collagen IV, α-SMA and fibronectin. * P <0.05 versus the TGF-β1 group. Bar graphs represent three independent experiments, each performed in triplicates. (B) Western blot analyses for the protein expression of collagen IV, α-SMA and fibronectin. (C) Protein levels were quantified by densitometry and normalized to the expression of GAPDH from three independent experiments. * P <0.05 versus the TGF-β1 group. (D-H) HK-2 cells were transfected with an siRNA against Brd4 or a negative control siRNA (si-NC) for 48 h or pretreated with 0.5 μM JQ1 for 1 h, and then treated with TGF-β1 (10 ng/mL) for 24 h. (D) Real-time PCR analyses for the mRNA expression of collagen IV, α-SMA and fibronectin in the indicated group. Bar graphs represent three independent experiments, each performed in triplicates. * P <0.05 versus TGF-β1; # P <0.05 versus si-NC. (E) Western blot analyses for the protein expression of collagen IV, α-SMA, and fibronectin in the indicated group. (F) Protein levels were quantified by densitometry and normalized to the expression of GAPDH from three independent experiments. * P <0.05 versus TGF-β1; # P <0.05 versus si-NC. (G) H 2 O 2 production in HK-2 cells in the indicated groups. Bar graphs represent three independent experiments, each performed in triplicates. * P <0.05 versus TGF-β1; # P <0.05 versus si-NC. (H) Representative images showing ROS stained with DCFH-DA dye in the indicated groups from three independent experiments.

Article Snippet: The rabbit anti-Brd4 antibody was supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Inhibition, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Negative Control, Staining

Journal: Redox Biology

Article Title: Brd4 inhibition attenuates unilateral ureteral obstruction-induced fibrosis by blocking TGF-β-mediated Nox4 expression

doi: 10.1016/j.redox.2016.12.031

Figure Lengend Snippet: Brd4 inhibition blocked Nox4-mediated ROS and fibrosis in HK-2 cells. (A) Representative bands of Western blot analyses for the expression of Nox4, collagen IV, α-SMA, and fibronectin in the presence of TGF-β1 or siRNA against Nox4 from three independent experiments. (B) H 2 O 2 production measured by Amplex Red in HK-2 cells transfected with siRNA against Nox4 or a negative control siRNA in the presence or absence of TGF-β1. Bar graphs represent three independent experiments, each performed in triplicates. * P <0.05 versus si-NC. (C) Real-time PCR analyses for the mRNA expression of Nox4 under the condition indicated from three independent experiments, each performed in triplicates. * P <0.05 versus TGF-β1; # P <0.05 versus si-NC. (D) Western Blot analyses for the protein expression of Nox4 and bar graph quantification as indicated from three independent experiments. * P <0.05 versus TGF-β1; # P <0.05 versus si-NC. (E-F) HK-2 cells were treated with TGF-β1 (10 ng/mL) for 24 h in the presence or absence of JQ1. TGF-β1+JQ1-treated HK-2 cells were then infected with adenovirus carrying the human Nox4 for 48 h. (E) Representative Western blot analyses of collagen IV, α-SMA and fibronectin in the indicated groups. (F) H 2 O 2 production measured by Amplex Red in HK-2 cells in the indicated groups. Bar graphs represent three independent experiments, each performed in triplicates. * P <0.05 versus the TGF-β1+JQ1 group.

Article Snippet: The rabbit anti-Brd4 antibody was supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Inhibition, Western Blot, Expressing, Transfection, Negative Control, Real-time Polymerase Chain Reaction, Infection

Journal: Redox Biology

Article Title: Brd4 inhibition attenuates unilateral ureteral obstruction-induced fibrosis by blocking TGF-β-mediated Nox4 expression

doi: 10.1016/j.redox.2016.12.031

Figure Lengend Snippet: Brd4 regulated Nox4 expression via the Smad and ERK pathways. (A) Western blot analyses for the protein expression of Smad3 and phosphorylated Smad3 in the indicated groups and quantitative analysis of Smad3 phosphorylation. * P <0.05 versus TGF-β1, # P <0.05 versus si-NC. (B) Western blot analyses for the protein expression of ERK1/2 and phosphorylated ERK1/2 in the indicated groups and quantitative analysis of ERK1/2 phosphorylation. * P <0.05 versus TGF-β1, # P <0.05 versus si-NC. (C) HK-2 cells were pretreated with either JQ1 (0.5 μM), SIS3 (Smad3 inhibitor, 10 μM) or U0126 (ERK1/2 inhibitor,10 μM) for 1 h and then treated with TGF-β1 (10 ng/mL) for 24 h. Western blot analyses for the protein expression of Nox4 in the indicated groups and quantification. * P <0.05 versus TGF-β1. (A-C) Each Western blot analysis is from three independent experiments. (D) Luciferase assay of Nox4 promoter activity in the presence of JQ1 or Brd4 knockdown with siRNA from three independent experiments, each performed in six replicates. * P <0.05 versus TGF-β1, # P <0.05 versus si-NC.

Article Snippet: The rabbit anti-Brd4 antibody was supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Phospho-proteomics, Luciferase, Activity Assay, Knockdown

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: (A) Schematic diagram depicting different types of alternatively spliced events (left). The bar graph (right) shows the distribution of alternatively spliced events among those that are differentially spliced in total thymus in BRD4 knock-out versus wild type (WT) (FDR < 0.05). Comparison of splicing events between WT and BRD4-deficient cells was based on transcripts expressed in both. (B) Developmental stages in thymocyte differentiation, DN (CD4, CD8 DN), ISP (CD8+ ISP), DP (CD4, CD8 DP), CD4, and CD8 single-positive thymocytes are shown. Arched arrows denote level of proliferative activity in DN and ISP thymocytes. (C) Bar graph showing the total number of differentially spliced events in the different thymocyte subpopulations in BRD4 knock-out versus wild-type thymus (FDR < 0.05), derived from RNA-seq analysis. BRD4 was conditionally deleted in DN thymocytes by LCK-Cre (Gegonne et al., 2018). Comparison of splicing events between WT and BRD4-deficient cells was based on transcripts expressed in both. See also Figure S1.

Article Snippet: Rabbit polyclonal anti- BRD4 [Epitope BRD4 C-terminal peptide (CFQSDLLSIFEENLF)] , Covance (Cutom antibody synthesis); Dey et al., 2000 , N/A.

Techniques: Knock-Out, Comparison, Activity Assay, Derivative Assay, RNA Sequencing

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: Summary of Immune Relevant Genes Whose Patterns of Splicing Are Altered by BRD4 Deficiency and the Thymocyte Subset in which the Alteration Occurs

Article Snippet: Rabbit polyclonal anti- BRD4 [Epitope BRD4 C-terminal peptide (CFQSDLLSIFEENLF)] , Covance (Cutom antibody synthesis); Dey et al., 2000 , N/A.

Techniques: Binding Assay

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: RNA from the different thymocyte subpopulations was subjected to RT-PCR for the indicated genes: CD45 (A and B), Arhgef1 (C and D), and Picalm (E and F). (A, C, and E) Upper panels: schematic diagrams depicting partial gene structure of the alternatively spliced genes CD45 (A), Arhgef1 (C), and Picalm (E). Rectangular boxes represent the exons, and the horizontal straight lines connecting the boxes represent the introns; the numbers below the boxes refer to the exon number of the gene, and numbers inside the boxes refer to the length of the exons; the numbers within the terminal exons do not refer to the actual exon length but the length amplifiable by the RT-PCR primers. The arrow heads show the approximate positions of the RT-PCR primers; boxes with hashed lines show the alternative exons; and curved lines connecting the boxes depict the splicing pattern. WT and KO refer to the splicing pattern prevalent in either the wild-type or knock-out thymocytes as determined by RNA-seq analysis. Lower panels: ethidium bromide stained agarose gels showing RT-PCR products derived from total RNA from BRD4 WT and KO thymocytes. (B, D, and F) Bar graphs of the RT-PCR results for CD45 (B), Arhgef1 (D), and Picalm (F). The ratios A/A+B (ratio of included exon transcript/total transcripts) were used as measure of alternative splicing and represent the average of three separate RT-PCR analyses. #, p < 0.05, significant difference between WT subpopulations, relative to WT DN; *p < 0.05, significant difference between WT and KO for the specific subpopulation. See also Figure S2.

Article Snippet: Rabbit polyclonal anti- BRD4 [Epitope BRD4 C-terminal peptide (CFQSDLLSIFEENLF)] , Covance (Cutom antibody synthesis); Dey et al., 2000 , N/A.

Techniques: Reverse Transcription Polymerase Chain Reaction, Knock-Out, RNA Sequencing, Staining, Derivative Assay, Alternative Splicing

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: (A) Effect of JQ1 or dBET6 treatment on the binding of BRD4 across the gene. ALL, TSS+gene body+TTS+intergenic; TSS, transcription start site; Gene body, between TSS and TTS; TTS, transcription termination site; intergenic, all remaining sequences. The peak distribution, in the absence of treatment is as follows: TSS, 1123; gene body, 4065; TTS, 226; intergenic, 1827. (B) Bar graph showing the distribution of alternative splice events among the differentially spliced events in response to JQ1 treatment or dBET6 treatment in T-ALL cells. (C) Bar graph showing the fraction of alternative splice (AS) genes that also have BRD4 associated with them at the TSS (pkAS). The total number of BRD4 peaks detected at the TSS across the genome was 1123. (D) Bar graph showing the fraction of AS genes that are also differentially expressed (DE) in response to JQ1 or dBET6 treatment. p values for (C) and (D) were obtained using a hypergeometric test, which tests the probability that the frequency of AS genes derived from either DE genes (overlap) or genes with BRD4-bound TSS peaks is larger than expected from the population; a low p value suggests the enrichment of AS genes in either DE genes or genes with BRD4 TSS peaks. See also Figures S3 and S4.

Article Snippet: Rabbit polyclonal anti- BRD4 [Epitope BRD4 C-terminal peptide (CFQSDLLSIFEENLF)] , Covance (Cutom antibody synthesis); Dey et al., 2000 , N/A.

Techniques: Binding Assay, Derivative Assay

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: (A) Immunoblot of BRD4 immunoprecipitates from thymocyte nuclear extracts (with and without benzonase treatment) with indicated antibodies to splicing factors FUS, HnRNPL, and U1–70. The immunoprecipitates from a single extract were run on either a 6% gel to visualize BRD4 and Fus or on a 10% gel to visualize HnRNPL and U1–70. The values under the IP lanes indicate the enrichment of anti-BRD4 co-IP, relative to the IgG control. (B, left) Immunoblot of BRD4 immunoprecipitates from HeLa nuclear extracts with indicated antibodies to splicing factors FUS, HnRNPM, U1–70, and U1-A. (B, right) Immunoblot of FUS immunoprecipitates from HeLa nuclear extracts with indicated antibodies to BRD4 and splicing factors HnRNPM, U1–70, and U1-A. (C) Schematic representation of BRD4 and BRD4-deletion mutants. The coordinates of the mouse BRD4 mutations are as follows. WT BRD4, 1402 aa; DN, 722–1402 aa; ΔC, 1–699aa; ΔBD1, 146–1402 aa; ΔBD2+B, 1–349/599–1402 aa; ΔB, 1–502/549–1402 aa; ΔET, 1–600/684–1402 aa; ΔHAT, 1–1156/1198–1402 aa. (D) Immunoblots showing pull-down analysis of recombinant BRD4 with recombinant HnRNPM. rHnRNPM (0.25 μg) was pulled down with rflag-BRD4 (0.5 μg) immobilized on Flag beads. Immunoblots were with anti-HnRNPM (upper) and anti-BRD4 (lower). (E) Immunoblots showing pull-down analysis of recombinant BRD4 with recombinant FUS. rFUS (0.25 μg) was pulled down with rflag-BRD4 (0.5 μg) WT or equimolar amounts of N-terminal or C-terminal BRD4 truncation mutants immobilized on Flag beads. Immunoblots were with anti-FUS (upper) and anti-BRD4 (lower). (F) Binding of HnRNPM (left panel) and FUS (right panel) to BRD4 mutants was assessed in pull-down assays with rBRD4 immobilized on Flag beads and immunoblotting with appropriate antibodies. The results represent the average of two experiments. (G) Retention of FUS and HnRNPM to BRD4 mutants, relative to the WT, was quantified as the fraction of input and normalized to the extent of binding to BRD4 WT. All results are representative of at least two independent experiments. See also Figure S5.

Article Snippet: Rabbit polyclonal anti- BRD4 [Epitope BRD4 C-terminal peptide (CFQSDLLSIFEENLF)] , Covance (Cutom antibody synthesis); Dey et al., 2000 , N/A.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Control, Recombinant, Binding Assay

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: (A) Proximity ligation assays (PLAs) were performed on primary thymocytes with anti-BRD4 and the antibodies for the indicated splicing factors. The PLAs are all significantly above the single antibody controls (Figure S6C). (B) PLA was performed using anti-BRD4 and the antibodies for the indicated splicing factors on fixed HeLa cells that had been treated with JQ1 (500 nM)/ DMSO for 6 hr. There is no significant difference (p > 0.05) between the treated and control PLA samples for either HnRNPM or Fus; both PLAs are significantly above single antibody alone controls (Figure S6C). PLA interaction is shown in red; DAPI staining in blue. See also Figures S6C and S6D.

Article Snippet: Rabbit polyclonal anti- BRD4 [Epitope BRD4 C-terminal peptide (CFQSDLLSIFEENLF)] , Covance (Cutom antibody synthesis); Dey et al., 2000 , N/A.

Techniques: Ligation, Control, Staining

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: (A) Metagene profile of BRD4 and FUS CHIP datasets showing colocalization of BRD4 and FUS at the TSS. (B) Log2 enrichment of reads in genomic features along the metagene body. (C) Enrichment heatmap showing co-localization of BRD4 with FUS across the genome. (D) Genome browser views of DNAAF3, ROBO3, and MAN1A1, showing BRD4 and FUS co-localization around the TSS and gene body. See also Figures S6A and S6B.

Article Snippet: Rabbit polyclonal anti- BRD4 [Epitope BRD4 C-terminal peptide (CFQSDLLSIFEENLF)] , Covance (Cutom antibody synthesis); Dey et al., 2000 , N/A.

Techniques:

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti- BRD4 [Epitope BRD4 C-terminal peptide (CFQSDLLSIFEENLF)] , Covance (Cutom antibody synthesis); Dey et al., 2000 , N/A.

Techniques: Recombinant, In Situ, cDNA Synthesis, Plasmid Preparation, Mutagenesis, Clone Assay, Software, Sequencing, Alternative Splicing