Journal: Theranostics

Article Title: Divergent splicing factor SRSF1 signaling promotes inflammation post-CME: the SRSF1/ENPP3 axis acts via inhibition of BRD4 O-GlcNAcylation to enhance NF-κB activation and accelerate heart failure

doi: 10.7150/thno.115402

Figure Lengend Snippet: O-GlcNAcylation of BRD4 inhibited NF-κB p65-mediated transcription of pro-inflammatory cytokines. (A)&(B) The expression of BRD4 in OGD-exposed cardiomyocytes was detected by RT-qPCR and Western blotting. H9C2 and AC-16 cells were transfected with shBRD4, and then subjected to OGD. (C)&(D) RT-qPCR and Western blotting analysis of BRD4 mRNA and protein levels. (E)&(F) The mRNA levels and concentrations of TNF-α, IL-1β, and IL-6 were determined by RT-qPCR and ELISA. (G) The binding of NF-κB p65 to TNF-α, IL-1β, and IL-6 promoters was confirmed by dual-luciferase reporter assay. (H)&(I) Co-IP assay verified the exogenous and endogenous interplay between OGT and BRD4 proteins. (J) O-GlcNAcylation of BRD4 protein in OGD-stimulated cardiomyocytes was evaluated. (K) YinOYang database predicated the potential O-GlcNAc sites on BRD4. OGD-challenged H9C2 and AC-16 cells were transfected with BRD4 WT plasmid or BRD4 plasmids with mutant O-GlcNAc sites (BRD4-S484R, BRD4-S784R, and BRD4-T1212R). (L) O-GlcNAcylation of BRD4 protein in H9C2 and AC-16 cells was detected. (M) Concentrations of TNF-α, IL-1β, and IL-6 were detected by ELISA. (N) The interaction between NF-κB p65 and TNF-α, IL-1β, and IL-6 promoters was validated by dual-luciferase reporter assay. n=3 for A-N. Student's t test (for A, B) and one-way ANOVA (for C-G, M, N) were performed to analyze data. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The membranes were treated overnight at 4 °C with primary antibodies targeting SRSF1 (12929-2-AP, 1:1000, Proteintech), ENPP3 (A05615, 1:1000, Boster), BRD4 (M00123, 1:500, Boster), p65 (ab32536, 1:1000, Abcam), β-tubulin (ab6046, 1:1000, Abcam), and β-actin (AC038, 1:10000, ABclonal, Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Binding Assay, Luciferase, Reporter Assay, Co-Immunoprecipitation Assay, Plasmid Preparation, Mutagenesis

Journal: Theranostics

Article Title: Divergent splicing factor SRSF1 signaling promotes inflammation post-CME: the SRSF1/ENPP3 axis acts via inhibition of BRD4 O-GlcNAcylation to enhance NF-κB activation and accelerate heart failure

doi: 10.7150/thno.115402

Figure Lengend Snippet: ENPP3 contributed to inflammation by inhibiting O-GlcNAcylation of BRD4. H9C2 and AC-16 cells were transfected with shENPP3, followed by exposure to OGD. (A) ENPP3 and BRD4 protein levels were measured by Western blotting. (B) The O-GlcNAc level of BRD4 protein was assessed. (C) The production of TNF-α, IL-1β, and IL-6 was determined by ELISA. (D) Dual-luciferase reporter assay evaluated the binding of NF-κB p65 to TNF-α, IL-1β, and IL-6 promoters. n=3 for A-D. One-way ANOVA was performed to analyze data. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The membranes were treated overnight at 4 °C with primary antibodies targeting SRSF1 (12929-2-AP, 1:1000, Proteintech), ENPP3 (A05615, 1:1000, Boster), BRD4 (M00123, 1:500, Boster), p65 (ab32536, 1:1000, Abcam), β-tubulin (ab6046, 1:1000, Abcam), and β-actin (AC038, 1:10000, ABclonal, Wuhan, China).

Techniques: Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, Binding Assay

Journal: Theranostics

Article Title: Divergent splicing factor SRSF1 signaling promotes inflammation post-CME: the SRSF1/ENPP3 axis acts via inhibition of BRD4 O-GlcNAcylation to enhance NF-κB activation and accelerate heart failure

doi: 10.7150/thno.115402

Figure Lengend Snippet: SRSF1/ENPP3 axis suppressed BRD4 O-GlcNAcylation to promote inflammation in CME. The OGD-stimulated cardiomyocytes were transfected with shSRSF1, ENPP3 overexpression plasmid, or a combination of them. (A) ENPP3 mRNA and lncRNA ENPP3 expression levels were detected by RT-qPCR. (B) The protein abundance of ENPP3 and BRD4 was assessed by Western blotting. (C) The O-GlcNAc level of BRD4 was determined. (D) ELISA was carried out to measure TNF-α, IL-1β, and IL-6 concentrations. n=3 for A-D. One-way ANOVA was performed to analyze data. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The membranes were treated overnight at 4 °C with primary antibodies targeting SRSF1 (12929-2-AP, 1:1000, Proteintech), ENPP3 (A05615, 1:1000, Boster), BRD4 (M00123, 1:500, Boster), p65 (ab32536, 1:1000, Abcam), β-tubulin (ab6046, 1:1000, Abcam), and β-actin (AC038, 1:10000, ABclonal, Wuhan, China).

Techniques: Transfection, Over Expression, Plasmid Preparation, Expressing, Quantitative RT-PCR, Quantitative Proteomics, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: Divergent splicing factor SRSF1 signaling promotes inflammation post-CME: the SRSF1/ENPP3 axis acts via inhibition of BRD4 O-GlcNAcylation to enhance NF-κB activation and accelerate heart failure

doi: 10.7150/thno.115402

Figure Lengend Snippet: Myocardium-specific SRSF1 knockout alleviated CME-induced inflammation via inactivation of the ENPP3/BRD4/NF-κB pathway. SRSF1 flox/flox and SRSF1-KO rats were injected with microspheres into the left ventricle to induce CME. (A) LVEF, LVFS, LVEDd, and CO were detected to evaluate cardiac function. (B) The serum cTnl level in different groups was measured by ELISA. (C) Pathological alterations in myocardial tissues were observed by HE staining (scale bar = 100 μm). (D) Myocardial infarct size was measured by HBFP staining (scale bar = 100 μm). (E) SRSF1, ENPP3, and BRD4 expression in myocardial tissues was evaluated by immunohistochemical staining (scale bar = 100 μm). (F) The protein abundance of SRSF1, ENPP3, BRD4, p65, and O-GlcNAcylation of BRD4 was detected by Western blotting or Co-IP, respectively. (G) ELISA was carried out to measure TNF-α, IL-1β, and IL-6 concentrations. n=6 for A-G. ANOVA for repeated measurement (for A, B), and one-way ANOVA (for F, G) was performed to analyze data. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The membranes were treated overnight at 4 °C with primary antibodies targeting SRSF1 (12929-2-AP, 1:1000, Proteintech), ENPP3 (A05615, 1:1000, Boster), BRD4 (M00123, 1:500, Boster), p65 (ab32536, 1:1000, Abcam), β-tubulin (ab6046, 1:1000, Abcam), and β-actin (AC038, 1:10000, ABclonal, Wuhan, China).

Techniques: Knock-Out, Injection, Enzyme-linked Immunosorbent Assay, Staining, Expressing, Immunohistochemical staining, Quantitative Proteomics, Western Blot, Co-Immunoprecipitation 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) 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

Journal: Nature Communications

Article Title: The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice

doi: 10.1038/s41467-020-19532-z

Figure Lengend Snippet: a mAID-BRD4 or TOP2-mAC HCT116 cells expressing OsTIR1(F74G) were treated with 1 µM 5-Ph-IAA and sampled at the indicated time points. Proteins were separated and detected using a specific antibody. Tubulin was used as a loading control. We repeated this experiment twice and obtained similar results. b Experimental time-course diagram showing the xenograft assay. c Graph showing mAID-BRD4 xenograft tumour growth. Tumour volume was checked on the indicated days. Data are presented as mean values ± SD ( n = 5 animals). d Graph showing the tumour weight on day 14. mAID-BRD4 xenograft tumour was taken and weighed. Data are presented as mean values ± SD ( n = 5 animals, two-tailed t -test). e Graph showing TOP2A-mAC xenograft tumour growth. Tumour volume was checked on the indicated days. Data are presented as mean values ± SD ( n = 4 animals). f Graph showing the tumour weight on day 14. TOP2A-mAC xenograft tumour was taken and weighed. Data are presented as mean values ± SD ( n = 4 animals, two-tailed t -test).

Article Snippet: Primary antibodies: anti-OsTIR1 (MBL, #PD048), anti-mAID (MBL, #M214-3), anti-DHC1 (SantaCruz, #sc-9115), anti-SMC2 (Bethyl, #A300-058A-T), anti-CTCF (Bethyl, #A300-543-T), anti-POLR2A (Abcom, #ab817), anti-BRD4 (GeneTex, #GTX130586), anti-TOP2A (MBL, #M042-3S), anti-alpha-tubulin (MBL, #M175-3), anti-beta-tubulin (SIGMA-Aldrich, #T4026), anti-alpha-tubulin conjugated with rhodamine (Bio-Rad, 12004165).

Techniques: Expressing, Xenograft Assay, Two Tailed Test