Journal: Cell death & disease

Article Title: The BRD4-SRPK2-SRSF2 signal modulates the splicing efficiency of ACSL3 pre-mRNA and influences erastin-induced ferroptosis in osteosarcoma cells.

doi: 10.1038/s41419-023-06273-2

Figure Lengend Snippet: Fig. 1 BRD4 inhibition protects osteosarcoma cells from erastin-ferrop in vitro. A Relative protein expression levels of BRD4 tested by western blotting in SaoS2 and U2-OS cells. B Cell survival rate analysis through the MTT assay. Intracellular MDA (C) and Fe2+ (D) content tested by analytical kits. E lipid ROS of each group determined by the boron-dipyrromethene C-11 probe and flow cytometry. F Electron microscopic images of mitochondria and the percentage of the damaged mitochondria. Tukey–Kramer test of one-way ANOVA, *: P < 0.05; **: P < 0.01; ***: P < 0.005.

Article Snippet: Then, primary antibodies of BRD4 (Merck KGaA, Cat. No. PLA0227), ACSL3 (Merck KGaA, Cat. No. HPA071021), ACSL4 (Proteintech, Cat. No. 22401-1-AP), ACSL5 (Proteintech, Cat No. 15708-1-AP), ACSL6 (Abcam, Cat. No. ab229937), SRPK2 (Abcam, Cat. No. ab251113), p-SRPK2 (CST, Cat. No. 23708), mTOR (CST, Cat. No. 2983), p-mTOR (CST, Cat. No. 5536), p70 S6K (CST, Cat. No. 9202), p-p70 S6K (CST, Cat. No. 97596), and SRSF2 (Merck KGaA, Cat. No. HPA049905) were diluted to a working concentration and incubated overnight at 4 °C.

Techniques: Inhibition, In Vitro, Expressing, Western Blot, MTT Assay, Cytometry

Journal: Cell death & disease

Article Title: The BRD4-SRPK2-SRSF2 signal modulates the splicing efficiency of ACSL3 pre-mRNA and influences erastin-induced ferroptosis in osteosarcoma cells.

doi: 10.1038/s41419-023-06273-2

Figure Lengend Snippet: Fig. 2 BRD4 inhibition protects osteosarcoma cells from erastin-ferrop in vivo. A Flowchart guide for the animal experiments. B Subcutaneous tumors from individual mice. Growth curve (C) and weights (D) of subcutaneous tumors. E MDA content in tumors. F Prussian blue staining of ferric ions and probe hybridization of lipid ROS in tumor tissue slides. Dunnett’s test of one-way ANOVA, *: P < 0.05; **: P < 0.01; ***: P < 0.005; n.s.: no significance.

Article Snippet: Then, primary antibodies of BRD4 (Merck KGaA, Cat. No. PLA0227), ACSL3 (Merck KGaA, Cat. No. HPA071021), ACSL4 (Proteintech, Cat. No. 22401-1-AP), ACSL5 (Proteintech, Cat No. 15708-1-AP), ACSL6 (Abcam, Cat. No. ab229937), SRPK2 (Abcam, Cat. No. ab251113), p-SRPK2 (CST, Cat. No. 23708), mTOR (CST, Cat. No. 2983), p-mTOR (CST, Cat. No. 5536), p70 S6K (CST, Cat. No. 9202), p-p70 S6K (CST, Cat. No. 97596), and SRSF2 (Merck KGaA, Cat. No. HPA049905) were diluted to a working concentration and incubated overnight at 4 °C.

Techniques: Inhibition, In Vivo, Staining, Hybridization

Journal: Cell death & disease

Article Title: The BRD4-SRPK2-SRSF2 signal modulates the splicing efficiency of ACSL3 pre-mRNA and influences erastin-induced ferroptosis in osteosarcoma cells.

doi: 10.1038/s41419-023-06273-2

Figure Lengend Snippet: Fig. 3 The dual effects of BRD4 on ACSL3 expression and subcellular location. A Functional domains of the complete ACSL3 protein with its corresponding aa length and the alignment result of the aa sequences for nine potential isoforms of ACSL3 collected from the UniProt database. B The genomic structures of ACSL3 exon skipping events of TCGA and GTEx across reference gene model from the ExonskipDB database. C, E Relative protein levels detected by western blotting. D Relative mRNA levels detected by RT-qPCR. F RNA stability assessed by RNA digestibility tests. G Detection of splicing variants of ACSL3 via RT-qPCR and agarose gel electrophoresis. Dunnett’s test of one-way ANOVA, *: P < 0.05; **: P < 0.01; ***: P < 0.005. H Typical images of ACSL3 and mitochondrial localization captured by laser confocal microscopy.

Article Snippet: Then, primary antibodies of BRD4 (Merck KGaA, Cat. No. PLA0227), ACSL3 (Merck KGaA, Cat. No. HPA071021), ACSL4 (Proteintech, Cat. No. 22401-1-AP), ACSL5 (Proteintech, Cat No. 15708-1-AP), ACSL6 (Abcam, Cat. No. ab229937), SRPK2 (Abcam, Cat. No. ab251113), p-SRPK2 (CST, Cat. No. 23708), mTOR (CST, Cat. No. 2983), p-mTOR (CST, Cat. No. 5536), p70 S6K (CST, Cat. No. 9202), p-p70 S6K (CST, Cat. No. 97596), and SRSF2 (Merck KGaA, Cat. No. HPA049905) were diluted to a working concentration and incubated overnight at 4 °C.

Techniques: Expressing, Functional Assay, Western Blot, Quantitative RT-PCR, Agarose Gel Electrophoresis, Confocal Microscopy

Journal: Cell death & disease

Article Title: The BRD4-SRPK2-SRSF2 signal modulates the splicing efficiency of ACSL3 pre-mRNA and influences erastin-induced ferroptosis in osteosarcoma cells.

doi: 10.1038/s41419-023-06273-2

Figure Lengend Snippet: Fig. 5 The effects of BRD4 on erastin-ferrop are partly working through the ACSL3 pathway. A, G Cell survival rate. B, H Intracellular content of MDA. C, I Intracellular content of Fe2+. E Relative protein levels detected by western blotting. F Abundance analysis of intracellular arachidonic acid. D, J Positive rate of intracellular lipid ROS. Dunnett’s test of one-way ANOVA, *: P < 0.05; **: P < 0.01; ***: P < 0.005; n.s.: no significant.

Article Snippet: Then, primary antibodies of BRD4 (Merck KGaA, Cat. No. PLA0227), ACSL3 (Merck KGaA, Cat. No. HPA071021), ACSL4 (Proteintech, Cat. No. 22401-1-AP), ACSL5 (Proteintech, Cat No. 15708-1-AP), ACSL6 (Abcam, Cat. No. ab229937), SRPK2 (Abcam, Cat. No. ab251113), p-SRPK2 (CST, Cat. No. 23708), mTOR (CST, Cat. No. 2983), p-mTOR (CST, Cat. No. 5536), p70 S6K (CST, Cat. No. 9202), p-p70 S6K (CST, Cat. No. 97596), and SRSF2 (Merck KGaA, Cat. No. HPA049905) were diluted to a working concentration and incubated overnight at 4 °C.

Techniques: Western Blot

Journal: Cell death & disease

Article Title: The BRD4-SRPK2-SRSF2 signal modulates the splicing efficiency of ACSL3 pre-mRNA and influences erastin-induced ferroptosis in osteosarcoma cells.

doi: 10.1038/s41419-023-06273-2

Figure Lengend Snippet: Fig. 6 SRPK2 is recruited by BRD4 and binds to its CTD domain. A 28 splicesome-associated proteins that bind to BRD4 via Co-IP and MS assay. B Unique sequences of SRPK1 and SRPK2 that bound to BRD4 protein analyzed by MS. C Relative mRNA levels detected by RT-qPCR. Dunnett’s test of one-way ANOVA, *: P < 0.05; **: P < 0.01; ***: P < 0.005. D RNA stability assessed by RNA digestibility tests. E IF staining results of ACSL3 in cells, typical images taken with laser confocal microscopy. F Endogenous binding relationship of BRD4 and SRPK2 identified by Co-IP method. G Yeast hybrid system to verify the binding domain of BRD4 (BD1, BD2, ET, and CTD domains) to SRPK2.

Article Snippet: Then, primary antibodies of BRD4 (Merck KGaA, Cat. No. PLA0227), ACSL3 (Merck KGaA, Cat. No. HPA071021), ACSL4 (Proteintech, Cat. No. 22401-1-AP), ACSL5 (Proteintech, Cat No. 15708-1-AP), ACSL6 (Abcam, Cat. No. ab229937), SRPK2 (Abcam, Cat. No. ab251113), p-SRPK2 (CST, Cat. No. 23708), mTOR (CST, Cat. No. 2983), p-mTOR (CST, Cat. No. 5536), p70 S6K (CST, Cat. No. 9202), p-p70 S6K (CST, Cat. No. 97596), and SRSF2 (Merck KGaA, Cat. No. HPA049905) were diluted to a working concentration and incubated overnight at 4 °C.

Techniques: Co-Immunoprecipitation Assay, Quantitative RT-PCR, Staining, Confocal Microscopy, Binding Assay

Journal: Cell death & disease

Article Title: The BRD4-SRPK2-SRSF2 signal modulates the splicing efficiency of ACSL3 pre-mRNA and influences erastin-induced ferroptosis in osteosarcoma cells.

doi: 10.1038/s41419-023-06273-2

Figure Lengend Snippet: Fig. 7 BRD4 affects splicing efficiency of pre-mACSL3 through SRPK2. A, D Relative protein levels detected by western blotting. B The enrichment of ACSL3 mRNA in the immunoprecipitate product of anti-SRSF2 antibody via the RIP/ RT-qPCR assay. C Relative mRNA levels detected by RT-qPCR. E RNA stability assessed by RNA digestibility tests. Dunnett’s test of one-way ANOVA, *: P < 0.05; **: P < 0.01; ***: P < 0.005.

Article Snippet: Then, primary antibodies of BRD4 (Merck KGaA, Cat. No. PLA0227), ACSL3 (Merck KGaA, Cat. No. HPA071021), ACSL4 (Proteintech, Cat. No. 22401-1-AP), ACSL5 (Proteintech, Cat No. 15708-1-AP), ACSL6 (Abcam, Cat. No. ab229937), SRPK2 (Abcam, Cat. No. ab251113), p-SRPK2 (CST, Cat. No. 23708), mTOR (CST, Cat. No. 2983), p-mTOR (CST, Cat. No. 5536), p70 S6K (CST, Cat. No. 9202), p-p70 S6K (CST, Cat. No. 97596), and SRSF2 (Merck KGaA, Cat. No. HPA049905) were diluted to a working concentration and incubated overnight at 4 °C.

Techniques: Western Blot, Quantitative RT-PCR

Journal: Cell death & disease

Article Title: The BRD4-SRPK2-SRSF2 signal modulates the splicing efficiency of ACSL3 pre-mRNA and influences erastin-induced ferroptosis in osteosarcoma cells.

doi: 10.1038/s41419-023-06273-2

Figure Lengend Snippet: Fig. 8 Correlation scatter plot between BRD4, SRPK2, SRSF2 and ACSL3 in osteosarcoma tissue. A Data from the GEO database (n = 18, Bivariate correlation analysis, R ≤0.4: low correlation; R å 0.4: middle correlation; P < 0.05: significant). B Intra- patient variation in diversity index (n = 10, Wilcoxon signed-rank test). C Schematic diagram of BRD4/SRPK2/SRSF2 axis in pre-mACSL3 splicing and expression.

Article Snippet: Then, primary antibodies of BRD4 (Merck KGaA, Cat. No. PLA0227), ACSL3 (Merck KGaA, Cat. No. HPA071021), ACSL4 (Proteintech, Cat. No. 22401-1-AP), ACSL5 (Proteintech, Cat No. 15708-1-AP), ACSL6 (Abcam, Cat. No. ab229937), SRPK2 (Abcam, Cat. No. ab251113), p-SRPK2 (CST, Cat. No. 23708), mTOR (CST, Cat. No. 2983), p-mTOR (CST, Cat. No. 5536), p70 S6K (CST, Cat. No. 9202), p-p70 S6K (CST, Cat. No. 97596), and SRSF2 (Merck KGaA, Cat. No. HPA049905) were diluted to a working concentration and incubated overnight at 4 °C.

Techniques: Expressing

Journal: bioRxiv

Article Title: BRD4 represses developmental and neuronal genes through interactions with polycomb complexes

doi: 10.64898/2026.01.31.702994

Figure Lengend Snippet: a ) Dot plots showing log2 fold enrichment of BRD proteins in the proximal interactome (Turbo-ID) for PRC1 and PRC2 proteins from mouse embryonic stem cells (mESCs), data from . The size of the circle represents the log2 fold enrichment in BRD4 IP relative to IgG control. b ) Like (a) but for enrichment of PRC proteins in BRD4 immunoprecipitation from K562 cells, data from , . The size of the circle represents the t-test difference between the BRD4 IP and the IgG control. c) Immunoblots of endogenous BRD4 IP in H9 hESCs using antibodies that recognise both short and long BRD4 isoforms, with antibodies detecting RING1B, CBX7, CBX4, H3K27ac, H3K23ac, H3K27me3, along with reverse IP with RING1B and MGA antibodies followed by immunoblots for BRD4 and H3K27me3. d ) Immunoblots of GFP-trap co-immunoprecipitation of GFP-BRD4 long isoform (GFP-BRD4L) with Flag-tagged E2F6 and L3MBTL2, HA-tagged EED and EZH2. Immunoblots for β-ACTIN served as controls, e ) Heatmap of CUT&Tag for BRD4, EED, H3K23ac and ChIP-seq data for H3K14ac and RING1B, at active (H3K4me3+), bivalent (H3K4me3+/H3K27me3+) and PRC2 repressed promoters (H3K27me3+). f ) AlphaScreen counts titration of BRD4-BD1 and -BD2 interaction with H3K14ac/23ac showing that only BRD4-BD2 interacts with H3K14ac/23ac. Normalized average alpha counts of three replicates were set relative to the highest WT. g) Immunoblots of biotinylated H3K14/K23ac pulldown for N-terminal His-FLAG tagged BRD4 (N-terminal 412 amino acids), in the presence of increasing concentration of iBET-BD2 (iBD2).

Article Snippet: 1 μg of biotinylated histone H3K14ac/H3K23ac peptide (Cayman Chemicals, Cat. 27520-250ug-CAY) was incubated with 10 μL of streptavidin magnetic beads (Invitrogen 656-01) in 300 μL of binding buffer (50 mM Tris, pH 7.5, 200 mM NaCl and 0.1% NP-40, proteinase inhibitor cocktail) and rotated at room temperature for 30 min. At the same time, FLAG-His tagged BRD4 N -terminal domain containing BD1 and BD2 (E49-E460) (MedChemExpress Cat# HY-P7846), inhibitor of iBET-BD2 (Cayman Chemical Cat# CAY31766), or DMSO were added to the binding buffer on ice.

Techniques: Control, Immunoprecipitation, Western Blot, ChIP-sequencing, Amplified Luminescent Proximity Homogenous Assay, Titration, Concentration Assay

Journal: bioRxiv

Article Title: BRD4 represses developmental and neuronal genes through interactions with polycomb complexes

doi: 10.64898/2026.01.31.702994

Figure Lengend Snippet: a ) Heatmap showing BRD4 signal (CPM) for WT and BRD4 BD2 mut1 at protein-coding genes and active enhancers of hESCs. b ) Scatter plot comparing log2 fold change (log2 FC) values for BRD4 BD2-Mut1/WT (X-axis) against BRD4 dTAG/DMSO (Y-axis) conditions. GSEA GO-biological process enrichment lists for genes that are commonly up (red) and down (blue) regulated in both conditions (right). c ) Representative genome browser snapshot displaying signals for RNA-seq WT, BRD4-mutant1, DMSO and dTAGV-1 along with MAX, BRD4, H3K27me3 and H3K4me3. For CUT&Tag (BRD2,3,4, H3K4me3, H3K27me3) and CUT&Run (EED, ser5 Pol-II), the signal is compared as CPM and MAX as ChIP-seq signal from ChIP-atlas. d) Heatmaps displaying H3K27me3 and H3K4me3 ChIP-seq signals along with RNA-seq normalized counts at bivalent genes in WT-H9 and H9-derived BRD4 BD2 mut1 neurons. e ) MA plot illustrating differential gene expression in BRD4 BD2 mut1 compared to WT neurons. Significantly up- and down-regulated bivalent and non-bivalent genes are highlighted in red and blue, respectively. The number of differentially expressed genes with a log2 fold change of 1 and an adjusted p-value of <0.05 is indicated (right). f ) Genome browser tracks showing ChIP-seq data for bivalent histone modifications (H3K4me3 and H3K27me3), fold change over input and RNA-seq (RPKM) for neuronal genes.

Article Snippet: 1 μg of biotinylated histone H3K14ac/H3K23ac peptide (Cayman Chemicals, Cat. 27520-250ug-CAY) was incubated with 10 μL of streptavidin magnetic beads (Invitrogen 656-01) in 300 μL of binding buffer (50 mM Tris, pH 7.5, 200 mM NaCl and 0.1% NP-40, proteinase inhibitor cocktail) and rotated at room temperature for 30 min. At the same time, FLAG-His tagged BRD4 N -terminal domain containing BD1 and BD2 (E49-E460) (MedChemExpress Cat# HY-P7846), inhibitor of iBET-BD2 (Cayman Chemical Cat# CAY31766), or DMSO were added to the binding buffer on ice.

Techniques: RNA Sequencing, ChIP-sequencing, Derivative Assay, Gene Expression

Journal: bioRxiv

Article Title: BRD4 represses developmental and neuronal genes through interactions with polycomb complexes

doi: 10.64898/2026.01.31.702994

Figure Lengend Snippet: a) Schematic representation of the protocol used to generate unguided neuronal organoids (UNOs), with images of UNO WT at 5,8, and 41 days. b ) Immunofluorescence images of UNOs at day 41 stained for markers of neuronal progenitor (SOX2), post-mitotic early neurons (TUJ1), scale bars: 100 μm. c ) MA plot for RNA-seq data illustrating differentially expressed genes in day 41 UNOs following 20 hours of BRD4 PROTAC (ZxH) treatment (n=3 independent organoids). d) Geneontology (GO) enrichment analyses of up- and down-regulated genes. e ) Genome browser tracks for normalized reads at TSS for pseudo bulk scCUT&Tag and bulk RNA-seq for immediate early genes (IEGs) upon 20 h BRD4 PROTAC in UNOs (data from (c)). f) UMAP plots stratified by genotype show the annotated cell lineages: WT, BRD4 BD2 mut2, and BRD4 BD2 mut3. Cell clusters are identified by colour, illustrating the contribution of each genotype to specific lineages, such as Glutamatergic, GABAnergic, optic vesicle, and RPE. g) Stacked bar charts for 41-day and 63-day UNOs, detailing the percentage of cells for each annotated cell type across the WT, BRD4 BD2 mut2, and BRD4 BD2 mut3 UNOs. h) Representative bright-field microscopy images of 41-day UNOs, Scale bar=1mm (rest of the images in source file). i) Dot plots showing the average expression level (Z scores) and percentage of cells expressed in Glutamatergic, Diencephalic-1(pink in UMAP), and Diencephalic-2(blue in UMAP), and G2M clusters for bivalent genes that showed significant differential expression in the scRNA-seq data in BRD4-BD2 mut1 and BRD4-BD2 mut2 UNOs.

Article Snippet: 1 μg of biotinylated histone H3K14ac/H3K23ac peptide (Cayman Chemicals, Cat. 27520-250ug-CAY) was incubated with 10 μL of streptavidin magnetic beads (Invitrogen 656-01) in 300 μL of binding buffer (50 mM Tris, pH 7.5, 200 mM NaCl and 0.1% NP-40, proteinase inhibitor cocktail) and rotated at room temperature for 30 min. At the same time, FLAG-His tagged BRD4 N -terminal domain containing BD1 and BD2 (E49-E460) (MedChemExpress Cat# HY-P7846), inhibitor of iBET-BD2 (Cayman Chemical Cat# CAY31766), or DMSO were added to the binding buffer on ice.

Techniques: Immunofluorescence, Staining, RNA Sequencing, Microscopy, Expressing, Quantitative Proteomics

Journal: bioRxiv

Article Title: BRD4 represses developmental and neuronal genes through interactions with polycomb complexes

doi: 10.64898/2026.01.31.702994

Figure Lengend Snippet: a) UMAP plots show the distribution of single-cell ATAC sequencing (scATAC-seq) data clustered by genotypes WT and BRD4 BD2 mut2 and annotated by cell lineage for WT and BRD4 BD2 mut2. b ) Z-scores (high scores in red and low scores are in blue) showing top transcription factor motifs enriched at Diencephalic, Glutamatergic, G2M and GABAnergic lineages across scATACseq peaks, which are gained in BRD4 BD2 mut 2 UNO compared to WT control. The complete list of enriched TFs is in the source data table.

Article Snippet: 1 μg of biotinylated histone H3K14ac/H3K23ac peptide (Cayman Chemicals, Cat. 27520-250ug-CAY) was incubated with 10 μL of streptavidin magnetic beads (Invitrogen 656-01) in 300 μL of binding buffer (50 mM Tris, pH 7.5, 200 mM NaCl and 0.1% NP-40, proteinase inhibitor cocktail) and rotated at room temperature for 30 min. At the same time, FLAG-His tagged BRD4 N -terminal domain containing BD1 and BD2 (E49-E460) (MedChemExpress Cat# HY-P7846), inhibitor of iBET-BD2 (Cayman Chemical Cat# CAY31766), or DMSO were added to the binding buffer on ice.

Techniques: Sequencing, Control

Journal: Nature Communications

Article Title: Identification of a SNAI1 enhancer RNA that drives cancer cell plasticity

doi: 10.1038/s41467-025-58032-w

Figure Lengend Snippet: a The interaction between SNAI1e and BRD4 in MDA‐MB‐231 cells was analyzed by RNA immunoprecipitation (RIP). YTHDC1 served as a negative control. RT-qPCR was performed to detect SNAI1e expression in immunoprecipitants from MDA‐MB‐231 cells. The results are expressed as mean ± SD from three independent experiments. b The interaction between SNAI1e and BRD4 in MDA-MB-231 cells was analyzed by RNA pull-down. Western blotting analysis was performed to detect FLAG expression in whole-cell lysates (Input) and immunoprecipitants (IP). LETS1 and SNAI1e-AS served as negative controls. The RNA amounts used for pull-down were evaluated by agarose gel electrophoresis. c The interaction between SNAI1e truncation mutants and BRD4 in MDA-MB-231 cells was analyzed by RNA pull-down. Western blotting analysis was performed to detect FLAG expression in whole-cell lysates (Input) and immunoprecipitants (IP). The RNA amounts used for pull-down were evaluated by agarose gel electrophoresis. d Schematic representation of full-length (FL) BRD4 and the truncation mutants tested. e The interactions between SNAI1e and BRD4 FL or the truncation mutants in MDA-MB-231 cells were analyzed by RNA pull-down. SNAI1e-AS , antisense SNAI1e ; SNAI1e-S , sense SNAI1e . Western blotting analysis was performed to detect FLAG expression in whole-cell lysates (Input) and immunoprecipitants (IP). The RNA amounts used for pull-down were evaluated by agarose gel electrophoresis. f The direct interaction between SNAI1e and the FLAG-BRD4 BD1/2 recombinant protein was analyzed by in vitro RIP. The results are expressed as mean ± SD from three independent experiments. The FLAG-tagged proteins in immunoprecipitants were evaluated by western blotting. g The direct interaction between SNAI1e and the recombinant FLAG-BRD4 BD1/2 protein was analyzed by in vitro RNA pull-down. Western blotting analysis was performed to detect FLAG expression in whole-cell lysates (Input) and immunoprecipitants (IP). The RNA amounts used for pull-down were evaluated by agarose gel electrophoresis. Significance was calculated by using one-way ANOVA followed by Dunnett’s ( a ) and Tukey’s ( f ) multiple comparisons test. Data are representative of at least three ( b , c , e , g ) independent experiments with similar results. Co.vec empty control vector.

Article Snippet: For in vitro RIP, 10 pmol of in vitro-transcribed SNAI1e was incubated with 2 μg recombinant FLAG-SMURF2 protein (Sigma‒Aldrich; SRP0228) or FLAG-BRD4 BD1/2 recombinant protein (R&D systems; SP-600) for 16 h at 4 °C as described previously .

Techniques: RNA Immunoprecipitation, Negative Control, Quantitative RT-PCR, Expressing, Western Blot, Agarose Gel Electrophoresis, Recombinant, In Vitro, Control, Plasmid Preparation

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 sections received overnight incubation with primary antibodies SRSF1 (12929-2-AP, 1:50, Proteintech, Wuhan, China), ENPP3 (A05615, 1:100, Boster, CA, USA), or BRD4 (M00123, 1:50, Boster) at 4 °C.

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 sections received overnight incubation with primary antibodies SRSF1 (12929-2-AP, 1:50, Proteintech, Wuhan, China), ENPP3 (A05615, 1:100, Boster, CA, USA), or BRD4 (M00123, 1:50, Boster) at 4 °C.

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 sections received overnight incubation with primary antibodies SRSF1 (12929-2-AP, 1:50, Proteintech, Wuhan, China), ENPP3 (A05615, 1:100, Boster, CA, USA), or BRD4 (M00123, 1:50, Boster) at 4 °C.

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 sections received overnight incubation with primary antibodies SRSF1 (12929-2-AP, 1:50, Proteintech, Wuhan, China), ENPP3 (A05615, 1:100, Boster, CA, USA), or BRD4 (M00123, 1:50, Boster) at 4 °C.

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