Journal: Frontiers in Immunology

Article Title: JQ1 as a BRD4 Inhibitor Blocks Inflammatory Pyroptosis-Related Acute Colon Injury Induced by LPS

doi: 10.3389/fimmu.2021.609319

Figure Lengend Snippet: Upregulated expression of BRD4 in endotoxemia mice colon with tight junction barrier injury. (A) HE staining and relative pathological scores of colon tissues from control mice (NS, normal saline, i.p.), and endotoxemia mice which were treated with LPS for 24 h (10 mg/kg, i.p.). Magnification (400×) bar represents 50 μm in all panels. (B) Western blot showed the reduced expression of tight junction protein occludin and ZO1 in colon of endotoxemia mice compared to saline-treated mice. (C) BRD4 protein level was upregulated in colon of endotoxemia mice. * p < 0.05, ** p < 0.01, n = 5/group, two-tailed unpaired Student’s t-test.

Article Snippet: After SDS-PAGE and transmembrane process, membranes were incubated with primary antibodies specific for the following proteins: BRD4 (Abclonal, A2249), occludin (Proteintech, 27260-1-AP), ZO1 (Proteintech, 21773-1-AP), IL6 (Proteintech, 21865-1-AP), IL18 (Proteintech, 10663-1-AP), IL1β (Proteintech, 16806-1-AP), p-NF κB (CST, 3033), NF κB (CST, 8242S), NLRP3 (CST, 15101S), ASC (Santan Cruze, sc-514414), cleaved-caspase 1 (Santan Cruze, sc56036), GSDMD (Abclonal, A18281), cleaved-N-terminal GSDME (Abcam, ab222407), GSDMA (Bioss, bs-16331R).

Techniques: Expressing, Staining, Western Blot, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: JQ1 as a BRD4 Inhibitor Blocks Inflammatory Pyroptosis-Related Acute Colon Injury Induced by LPS

doi: 10.3389/fimmu.2021.609319

Figure Lengend Snippet: JQ1 as a novel BRD4 inhibitor repaired the tight junction damage of endotoxemia colon. Mice were injected with JQ1 (50 mg/kg, i.p.) 1 h before LPS (10 mg/kg) injection. NS group was treated with DMSO, which was the solvent of JQ1. Colon were harvested 24 h after LPS injection for western blot, HE staining, and IF assay. (A) JQ1 blocked BRD4 protein level in colon of endotoxemia mice. (B) HE staining and relative pathological scores showed pretreatment with BRD4 inhibitor JQ1 reversed the injury of colon mucosal layer. 400×, bar value was 50 μm. (C) Colonic permeability assay indicated the protection function of JQ1 in colonic barriers. (D) Expression of tight junction protein occludin and ZO1 in endotoxemia colon were detected by western blot. (E) Immunofluorescence of occludin also indicated the protection role of JQ1 in endotoxemia colon. Bar value was 50 μm. * p < 0.05, ** p < 0.01, *** p < 0.001, vs NS group. # p < 0.05, ## p < 0.01, ### p < 0.001, vs LPS group. n = 5/group, one-way ANOVA.

Article Snippet: After SDS-PAGE and transmembrane process, membranes were incubated with primary antibodies specific for the following proteins: BRD4 (Abclonal, A2249), occludin (Proteintech, 27260-1-AP), ZO1 (Proteintech, 21773-1-AP), IL6 (Proteintech, 21865-1-AP), IL18 (Proteintech, 10663-1-AP), IL1β (Proteintech, 16806-1-AP), p-NF κB (CST, 3033), NF κB (CST, 8242S), NLRP3 (CST, 15101S), ASC (Santan Cruze, sc-514414), cleaved-caspase 1 (Santan Cruze, sc56036), GSDMD (Abclonal, A18281), cleaved-N-terminal GSDME (Abcam, ab222407), GSDMA (Bioss, bs-16331R).

Techniques: Injection, Western Blot, Staining, Permeability, Expressing, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: JQ1 as a BRD4 Inhibitor Blocks Inflammatory Pyroptosis-Related Acute Colon Injury Induced by LPS

doi: 10.3389/fimmu.2021.609319

Figure Lengend Snippet: BRD4 inhibition by JQ1 blocked colon inflammation during endotoxemia. Results of Q-PCR (A) and western blot (B) showed the BRD4 inhibitor JQ1 could block the rising trend of inflammation cytokines IL6, IL18, and IL1β at gene level and protein level respectively. * p < 0.05, ** p < 0.01, vs NS group. # p < 0.05, ## p < 0.01, 0.01, ### p < 0.001, vs LPS group. n = 6/group, one-way ANOVA.

Article Snippet: After SDS-PAGE and transmembrane process, membranes were incubated with primary antibodies specific for the following proteins: BRD4 (Abclonal, A2249), occludin (Proteintech, 27260-1-AP), ZO1 (Proteintech, 21773-1-AP), IL6 (Proteintech, 21865-1-AP), IL18 (Proteintech, 10663-1-AP), IL1β (Proteintech, 16806-1-AP), p-NF κB (CST, 3033), NF κB (CST, 8242S), NLRP3 (CST, 15101S), ASC (Santan Cruze, sc-514414), cleaved-caspase 1 (Santan Cruze, sc56036), GSDMD (Abclonal, A18281), cleaved-N-terminal GSDME (Abcam, ab222407), GSDMA (Bioss, bs-16331R).

Techniques: Inhibition, Western Blot, Blocking Assay

Journal: Frontiers in Immunology

Article Title: JQ1 as a BRD4 Inhibitor Blocks Inflammatory Pyroptosis-Related Acute Colon Injury Induced by LPS

doi: 10.3389/fimmu.2021.609319

Figure Lengend Snippet: BRD4 activated phosphorylated NF κb and promoted the expression of NLRP3/ASC/Caspase 1 inflammasome complex in endotoxemia colon. (A) Western blot disclosed that phosphorylated NF κb was activated in endotoxemia colon, which was reversed by BRD4 inhibitor JQ1. (B, C) Pretreatment with JQ1 prevents gene and protein expression of the NLRP3/ASC/caspase1 inflammasome complex in endotoxemia colon. * p < 0.05, ** p < 0.01, vs NS group. # p < 0.05, ## p < 0.01, ### p < 0.001 vs LPS group. n = 6/group, one-way ANOVA.

Article Snippet: After SDS-PAGE and transmembrane process, membranes were incubated with primary antibodies specific for the following proteins: BRD4 (Abclonal, A2249), occludin (Proteintech, 27260-1-AP), ZO1 (Proteintech, 21773-1-AP), IL6 (Proteintech, 21865-1-AP), IL18 (Proteintech, 10663-1-AP), IL1β (Proteintech, 16806-1-AP), p-NF κB (CST, 3033), NF κB (CST, 8242S), NLRP3 (CST, 15101S), ASC (Santan Cruze, sc-514414), cleaved-caspase 1 (Santan Cruze, sc56036), GSDMD (Abclonal, A18281), cleaved-N-terminal GSDME (Abcam, ab222407), GSDMA (Bioss, bs-16331R).

Techniques: Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: JQ1 as a BRD4 Inhibitor Blocks Inflammatory Pyroptosis-Related Acute Colon Injury Induced by LPS

doi: 10.3389/fimmu.2021.609319

Figure Lengend Snippet: Inhibition of BRD4 blocked the expression of pyroptosis-related proteins in endotoxemia colon. In endotoxemia colon, pyroptosis-related proteins GSDMD, GSDME, and GSDMA with active forms were upregulated at gene (A) and protein level (B) , which were powerfully reversed by BRD4 inhibitor JQ1. * p < 0.05, ** p < 0.01, *** p < 0.001 vs NS group. # p < 0.05, ## p < 0.01, ### p < 0.001 vs LPS group. n = 6/group, one-way ANOVA.

Article Snippet: After SDS-PAGE and transmembrane process, membranes were incubated with primary antibodies specific for the following proteins: BRD4 (Abclonal, A2249), occludin (Proteintech, 27260-1-AP), ZO1 (Proteintech, 21773-1-AP), IL6 (Proteintech, 21865-1-AP), IL18 (Proteintech, 10663-1-AP), IL1β (Proteintech, 16806-1-AP), p-NF κB (CST, 3033), NF κB (CST, 8242S), NLRP3 (CST, 15101S), ASC (Santan Cruze, sc-514414), cleaved-caspase 1 (Santan Cruze, sc56036), GSDMD (Abclonal, A18281), cleaved-N-terminal GSDME (Abcam, ab222407), GSDMA (Bioss, bs-16331R).

Techniques: Inhibition, Expressing

Journal: Science advances

Article Title: Hypoxia deactivates epigenetic feedbacks via enzyme-derived clicking proteolysis-targeting chimeras.

doi: 10.1126/sciadv.abq2216

Figure Lengend Snippet: Fig. 1. Illustration of ENCTAC performances under hypoxic condition for selective degradation of BRD4 proteins.

Article Snippet: Primary antibodies against BRD4 (#13440; 1:1000 dilution factor), HIF-1α (#36169; 1:1000 dilution factor), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #5174; 1:1000 dilution factor) were purchased from Cell Signaling Technologies; c-Myc (#700648; 1:1000 dilution factor), PARP1 (#MA5-15031; 1:500 dilution factor), VEGF (#P802; 1:500 dilution factor), and CA-IX (#MA5-29076; 1:1000 dilution factor) were purchased from Thermo Fisher Scientific; BRD4 (ab243862; 1:1000 dilution factor) and β-tubulin (ab6046; 1:10,000 dilution factor) were purchased from Abcam.

Techniques:

Journal: Science advances

Article Title: Hypoxia deactivates epigenetic feedbacks via enzyme-derived clicking proteolysis-targeting chimeras.

doi: 10.1126/sciadv.abq2216

Figure Lengend Snippet: Fig. 2. Enzyme-derived click formation of heterobifunctional degraders of BRD4. (A) Chemical structure of GSH-responsive CRBN ligand (J266); GSH- and NTR- responsive CRBN ligand (JW4); cleaved J266; CBT-linked BRD4-targeting ligand (JQ1-CBT); click-induced BRD4 degrader (J252). (B) LC-MS spectra of NTR uncaging JW4 (10 μM) to form J266 at different time points in NTR (40 μg/ml) dissolved in phosphate-buffered saline (PBS; 10 mM) (pH 7.4). (C) Selectivity of JW4 toward a broad range of biological and chemical agents with or without NADH. (D) Time-dependent LC-MS spectra of click J252 formation under reducing reagent [tris(2-carbox- yethyl)phosphine (TCEP)] that cleave the J266 to induce cleaved J266 before the click reaction with JQ1-CBT in buffer solution. (E) Ratio of peak areas of click J252 and cleaved J266 to J266 over multiple time points in (D).

Article Snippet: Primary antibodies against BRD4 (#13440; 1:1000 dilution factor), HIF-1α (#36169; 1:1000 dilution factor), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #5174; 1:1000 dilution factor) were purchased from Cell Signaling Technologies; c-Myc (#700648; 1:1000 dilution factor), PARP1 (#MA5-15031; 1:500 dilution factor), VEGF (#P802; 1:500 dilution factor), and CA-IX (#MA5-29076; 1:1000 dilution factor) were purchased from Thermo Fisher Scientific; BRD4 (ab243862; 1:1000 dilution factor) and β-tubulin (ab6046; 1:10,000 dilution factor) were purchased from Abcam.

Techniques: Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Saline

Journal: Science advances

Article Title: Hypoxia deactivates epigenetic feedbacks via enzyme-derived clicking proteolysis-targeting chimeras.

doi: 10.1126/sciadv.abq2216

Figure Lengend Snippet: Fig. 3. Heterobifunctional degraders efficiently disrupt BRD4 protein. (A) Molecular dynamic simulation of J252 interacting with CRBN (gold) and BRD4 (blue) pro- teins. The position of the luciferin-based component shows no steric collision with BRD4 or CRBN. (snapshot at 1 ns). (B) Western blot analysis of BRD4 protein after treatment with J252 and ARV-825 in human embryonic kidney (HEK) 293T cells for 24 hours with indicated concentration and β-tubulin as internal controls.

Article Snippet: Primary antibodies against BRD4 (#13440; 1:1000 dilution factor), HIF-1α (#36169; 1:1000 dilution factor), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #5174; 1:1000 dilution factor) were purchased from Cell Signaling Technologies; c-Myc (#700648; 1:1000 dilution factor), PARP1 (#MA5-15031; 1:500 dilution factor), VEGF (#P802; 1:500 dilution factor), and CA-IX (#MA5-29076; 1:1000 dilution factor) were purchased from Thermo Fisher Scientific; BRD4 (ab243862; 1:1000 dilution factor) and β-tubulin (ab6046; 1:10,000 dilution factor) were purchased from Abcam.

Techniques: Western Blot, Concentration Assay

Journal: Science advances

Article Title: Hypoxia deactivates epigenetic feedbacks via enzyme-derived clicking proteolysis-targeting chimeras.

doi: 10.1126/sciadv.abq2216

Figure Lengend Snippet: Fig. 4. Hypoxia-activated degradation of epigenetic BRD4 protein. (A) Illustration of intracellular hypoxic enzyme uncaging, formation of the degrader (click J252), and degradation of BRD4. (B) Western blot analysis of BRD4 protein levels after treatments with GSH-cleavable J266 (6 hours) and JQ1-CBT (12 hours) at indicated con- centration and different time points; GSH- and NTR-responsive JW4 (6 hours) with JQ1-CBT (12 hours) at the indicated concentration and for a different time period under hypoxia in HEK-293T cells. (C) Analysis of BRD4 protein levels under the combination of different control conditions for 12 hours (the inhibitor bortezomib incubated with cells for 2 hours before the addition of JW4 and JQ1-CBT). (D) Western blot analysis of BRD4 protein levels after treatments with J266 (6 hours) and JQ1-CBT (12 hours) or JW4 (6 hours) and JQ1-CBT (12 hours) in time- and concentration-dependent examinations of HeLa cancer cells; analysis of BRD4 protein levels after treatments with JQ1 (12 hours) in a concentration-dependent manner of hypoxia HeLa cells; and JW4 (6 hours) and JQ1-CBT (12 hours) in normoxia HeLa cells. (E) Extended concentration of degraders demonstrating the hook effect during incubation under hypoxia (representative results from two to three biological replications). β-Tubulin was used as in- ternal controls. (F) BRD4 degradation level over varied concentration as indicated in (E). Values represent triplicate means ± SD, normalized to nontreated cells, and baseline-corrected using immunoblots.

Article Snippet: Primary antibodies against BRD4 (#13440; 1:1000 dilution factor), HIF-1α (#36169; 1:1000 dilution factor), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #5174; 1:1000 dilution factor) were purchased from Cell Signaling Technologies; c-Myc (#700648; 1:1000 dilution factor), PARP1 (#MA5-15031; 1:500 dilution factor), VEGF (#P802; 1:500 dilution factor), and CA-IX (#MA5-29076; 1:1000 dilution factor) were purchased from Thermo Fisher Scientific; BRD4 (ab243862; 1:1000 dilution factor) and β-tubulin (ab6046; 1:10,000 dilution factor) were purchased from Abcam.

Techniques: Western Blot, Concentration Assay, Control, Incubation

Journal: Science advances

Article Title: Hypoxia deactivates epigenetic feedbacks via enzyme-derived clicking proteolysis-targeting chimeras.

doi: 10.1126/sciadv.abq2216

Figure Lengend Snippet: Fig. 5. Hypoxic BRD4 degradation resulted in a change in the response of the cellular microenvironment and malfunction of cell growth. (A) Confocal imaging of HIF-1α immunostaining (green; λex = 488 nm and λem = 515/30 nm) after subjecting HeLa cells to the as-stated treatment. Nucleus was stained with Hoechst 33258 (blue; λex = 405 nm and λem = 460/50 nm), β-tubulin was stained with the fluorescent β-tubulin antibody (red; λex = 561 nm and λem = 617/20 nm). Scale bars, 40 μm. (B) Quantitative mean fluorescence intensity of HIF-1α after treatment and staining as indicated in (A). ka.u., kilo–arbitrary units. Values represent mean fluorescence intensity of three different cell areas ± SD. Western blot analysis of HIF-1α after treatment with different concentrations of JW4 and JQ1-CBT (C) or separated JW4, JQ1-CBT, JQ1, and J252 controls (12 hours) (D). (E) Western blot analysis of vascular endothelial growth factor (VEGF) and CA9 after hypoxia-activated ENCTAC treatment (12 hours). (F) Protein level of HIF-1α, VEGF, and CA9 after hypoxia ENCTAC (blue bar) and JQ1 inhibitor treatment (12 hours) (red bar). Values represent the average of duplicates and the range as error bars, normalized to nontreated cells, and baseline-corrected using immunoblots. (G) Immunoblot for c-Myc and glyceraldehyde-3-phosphate dehydro- genase (GAPDH) levels after hypoxia-activated ENCTAC treatment using JW4 and JQ1-CBT for 12 hours. (H) Immunoblot for poly(adenosine 5′-diphosphate–ribose) polymerase (PARP) cleavage and GAPDH levels after similar treatment condition as in (G). (I) Confocal imaging of apoptosis cell death staining with annexin V (AnnV)/propodium iodide (PI) [AnnV (green), λex = 488 nm and λem = 520/30 nm; PI (red), λex = 561 nm and λem = 590/30 nm]. Hypoxic cells without ENCTAC treatment as control. BF, bright field. Scale bars, 40 μm. (J) Flow cytometry of apoptosis/necrosis-stained HeLa cells under treatments with JW4, JQ1-CBT individually, or in com- bination of JW4 and JQ1-CBT (10 μM) (12 hours). Quarter 1 (Q1) indicates the relative percentage of necrosis cells, Q2 indicates late apoptosis cells, Q3 indicates early apoptosis cells, and Q4 indicates live cells.

Article Snippet: Primary antibodies against BRD4 (#13440; 1:1000 dilution factor), HIF-1α (#36169; 1:1000 dilution factor), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #5174; 1:1000 dilution factor) were purchased from Cell Signaling Technologies; c-Myc (#700648; 1:1000 dilution factor), PARP1 (#MA5-15031; 1:500 dilution factor), VEGF (#P802; 1:500 dilution factor), and CA-IX (#MA5-29076; 1:1000 dilution factor) were purchased from Thermo Fisher Scientific; BRD4 (ab243862; 1:1000 dilution factor) and β-tubulin (ab6046; 1:10,000 dilution factor) were purchased from Abcam.

Techniques: Imaging, Immunostaining, Staining, Fluorescence, Western Blot, Control, Flow Cytometry

Journal: Science advances

Article Title: Hypoxia deactivates epigenetic feedbacks via enzyme-derived clicking proteolysis-targeting chimeras.

doi: 10.1126/sciadv.abq2216

Figure Lengend Snippet: Fig. 6. In vivo degradation of BRD4 using ENCTACs to manipulate hypoxic zebrafish development. (A) Scheme of zebrafish early treatment with hypoxia-activated ENCTACs. (B) Western blot analysis of BRD4 level in hypoxia zebrafish after treatment with different concentrations of ENCTAC molecules. (C) Western blot analysis of HIF- 1α level in hypoxia zebrafish upon treatment with different concentrations of ENCTAC molecules or with inhibitor JQ1. (D) Bright-field images of zebrafish embryos under different conditions of indicated drug treatment at 36 hpf. Red arrowheads indicate yolk extension area. (E) Fluorescent imaging of zebrafish larvae phenotype with blood vessel trackers ( fli1:eGFPy1) in wild-type (WT), and von Hippel–Lindau (vhl) mutant (vhl−/−) larvae with and without ENCTAC treatment. Blood vessel tracker (green), λex = 488 nm and λem = 520/30 nm. Scale bar, 500 μm. (F) Fluorescent intensity spectra of blood vessel alignment along red line (I to IV) indicated in (E). (G) Statistical numbers of vascularization phenotypes in zebrafish larvae with and without ENCTAC treatments or with PROTAC, J252, or inhibitor JQ1 (10 μM).

Article Snippet: Primary antibodies against BRD4 (#13440; 1:1000 dilution factor), HIF-1α (#36169; 1:1000 dilution factor), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #5174; 1:1000 dilution factor) were purchased from Cell Signaling Technologies; c-Myc (#700648; 1:1000 dilution factor), PARP1 (#MA5-15031; 1:500 dilution factor), VEGF (#P802; 1:500 dilution factor), and CA-IX (#MA5-29076; 1:1000 dilution factor) were purchased from Thermo Fisher Scientific; BRD4 (ab243862; 1:1000 dilution factor) and β-tubulin (ab6046; 1:10,000 dilution factor) were purchased from Abcam.

Techniques: In Vivo, Western Blot, Imaging, Mutagenesis

Journal: Science advances

Article Title: Hypoxia deactivates epigenetic feedbacks via enzyme-derived clicking proteolysis-targeting chimeras.

doi: 10.1126/sciadv.abq2216

Figure Lengend Snippet: Fig. 7. Tumor inhibition by ENCTACs assisted BRD4 degradation. (A) Schematic diagram shows the treatment of ENCTACs in the mouse melanoma xenograft model. (B) Fluorescence image of NTR activity in solid tumor. Near-infrared fluorescence reporter was intratumorally injected, and the image was taken 30 min later. (C) Immu- noblot for BRD4, c-Myc, HIF-1α, and GAPDH in tumor lysates collected from mice treated with JW4 and JQ1-CBT (5 mg/kg) mixture, JQ1 (5 mg/kg) alone, or vehicle twice for 4 hours. (D) Representative pictures and quantitative analysis of tumor volume (means ± SD) of vehicle-treated mice (n = 3) or mice treated with JW4, JQ1-CBT mixture (5 mg/kg; n = 4), or JQ1-treated mice (n = 3) for 5 days. (E) Relative tumor growth rate under different treatments as indicated for 5 days. (F) Immunohistochemistry staining with a vascular marker CD31 (green) and nucleus marker 4′,6-diamidino-2-phenylindole (DAPI) (blue) of tumors subjected to ENCTACs, (+)-JQ1, or vehicle control. (G) Quantification of total vascular area in tumors subjected to ENCTACs, (+)-JQ1, or vehicle control.

Article Snippet: Primary antibodies against BRD4 (#13440; 1:1000 dilution factor), HIF-1α (#36169; 1:1000 dilution factor), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #5174; 1:1000 dilution factor) were purchased from Cell Signaling Technologies; c-Myc (#700648; 1:1000 dilution factor), PARP1 (#MA5-15031; 1:500 dilution factor), VEGF (#P802; 1:500 dilution factor), and CA-IX (#MA5-29076; 1:1000 dilution factor) were purchased from Thermo Fisher Scientific; BRD4 (ab243862; 1:1000 dilution factor) and β-tubulin (ab6046; 1:10,000 dilution factor) were purchased from Abcam.

Techniques: Inhibition, Fluorescence, Activity Assay, Injection, Immunohistochemistry, Staining, Marker, Control

Journal: Science advances

Article Title: Hypoxia deactivates epigenetic feedbacks via enzyme-derived clicking proteolysis-targeting chimeras.

doi: 10.1126/sciadv.abq2216

Figure Lengend Snippet: Fig. 8. Pharmacokinetic analysis and tumor inhibition studies of ENCTACs. (A and B) Plasma concentration–time profiles of JW4 (A) and JQ1-CBT (X = NH) (B) after intravenous dosing (5 mg/kg). (C) Schematic diagram for the treatment of ENCTACs in the xenograft mouse model with melanoma and HeLa tumor. (D) Representative images and quantitative analysis of tumor volume (means ± SD) of melanoma mice treated with vehicle (n = 4), JQ1 (5 mg/kg, n = 5), ARV-825 (5 mg/kg; n = 5), or JW4 + JQ1-CBT mixture (5 mg/kg; n = 5) for 6 days. (E) Western blot analysis of BRD4 levels in melanoma tumor after intravenous injection of vehicle, JQ1, ARV-825, or JW4 + JQ1-CBT (5 mg/kg). (F) Representative images and quantitative analysis of tumor volume (means ± SD) of HeLa tumor–bearing mice treated with vehicle control (n = 3), JQ1 (5 mg/kg, n = 3), ARV- 825 (5 mg/kg; n = 3), or JW4 + JQ1-CBT mixture (5 mg/kg; n = 3) for 8 days.

Article Snippet: Primary antibodies against BRD4 (#13440; 1:1000 dilution factor), HIF-1α (#36169; 1:1000 dilution factor), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #5174; 1:1000 dilution factor) were purchased from Cell Signaling Technologies; c-Myc (#700648; 1:1000 dilution factor), PARP1 (#MA5-15031; 1:500 dilution factor), VEGF (#P802; 1:500 dilution factor), and CA-IX (#MA5-29076; 1:1000 dilution factor) were purchased from Thermo Fisher Scientific; BRD4 (ab243862; 1:1000 dilution factor) and β-tubulin (ab6046; 1:10,000 dilution factor) were purchased from Abcam.

Techniques: Inhibition, Clinical Proteomics, Concentration Assay, Western Blot, Injection, Control

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: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: E2F1-BRD4 binding is lost with K117 monomethylated E2F1 in vitro . ( A ) Crystal structure of the human BRD4 BD1 (sandy-brown) in complex with an acetylated K117ac/K120ac E2F1 peptide (blue) (PDB 6ULS) showing the key interaction of K117ac with BD1. ( B ) Coomassie BB stained 12% SDS gel of the purified GST tagged truncated BRD4 (2-477 aa) protein including BD1 and BD2 (BD1/2) as well as the purified BD1 domain (2-220 aa). The GST-tagged BRD4 proteins are marked with asterisks. ( C ) Binding of the GST-BRD4 BD1/2 to modified E2F1 peptides. 15 aa long E2F1 peptides with different combinations of unmodified, acetylated, and methylated K117 and K120 were synthesized on peptide SPOT arrays. The sequence of each peptide is listed in the table. Peptide arrays were incubated with 5 nM GST-BRD4 BD1/2 and binding was detected using a GST-specific antibody. The bar diagram shows the binding of E2F1-BRD4 to K117ac/K120ac and K117me/K120ac observed in three independent experiments. The bars represent the averages. The P- value was determined by two flanked t ‐test with equal variance. ( D ) Same as in panel (C), but GST-BRD4 BD1 was used. Additional data are provdied in .

Article Snippet: PLA Duolink assays were performed according to the manufacturer’s instructions (Sigma) using antibodies against BRD4 (Bethyl, A700-004), E2F1 (SantaCruz, SC-251) and Flag (Sigma, F1804) overnight at 4°C.

Techniques: Binding Assay, In Vitro, Staining, SDS-Gel, Purification, Modification, Methylation, Synthesized, Sequencing, Incubation

Journal: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: E2F1-BRD4 binding is lost with K117 monomethylated E2F1 in cells. ( A ) GFP-tagged BRD4 (2-477) and Flag-E2F1 (2-437) were transfected into DU145 SETD6 WT and KO cells. The GFP-tagged BRD4 was purified by GFP-trap and analyzed by western-blot with an anti-GFP antibody. Co-purification of Flag-E2F1 was determined by anti-Flag antibody. Equal loading of cell lysate isolated from transfected DU145 SETD6 WT or KO was verified by western-blot analysis against β-actin, GFP, and Flag. ( B ) GFP-tagged BRD4 (2-477) and Flag-E2F1 (2-437) WT or K117R were transfected into in DU145 SETD6 KO cells. Some of the transfected cells were treated with JQ1-Bromodomain-Kac binding inhibitor (5 µM) or DMSO as control. GPF-trap and western-blot analysis was conducted as in panel A. (C–E) Interaction of BRD4 and E2F1 investigated by PLA. All experiments were conducted in DU145 cells. Exemplary microscopy images are shown. Scale bar: 10 µm. PLA signal quantification (PLA dots per nucleus, AU) for each sample is shown on the right. Statistical analysis was performed using Student’s t -test in GraphPad (**** P < .0001). ( C ) Interaction of endogenous BRD4 and Flag-E2F1 in the absence and the presence of the SAHA deacetylase inhibitor (20 µM) for 5 h (Flag-E2F1). Negative control (Neg) refers to reaction conducted without addition of Flag primary antibody. The interaction of BRD4 and E2F1 was detected and it was shown to be stimulated by increasing acetylation levels after SAHA treatment. Number of analyzed cells: 183, 132, 223. ( D ) Detection of the interaction of endogenous BRD4 and endogenous E2F1 in the presence of 40 µM SAHA for 5 h in DU145 cells (Control) and SETD6 KO cells (KO1 and KO2). Negative control (Negative) refers to reaction conducted without addition of E2F1 primary antibody. Number of analyzed cells: 67, 236, 104, 91. ( E ) Interaction of endogenous BRD4 and endogenous E2F1 in the presence of 40 µM SAHA for 5 h in DU145 cells and with overexpression of GFP (GFP empty) or GFP-SETD6 (GFP-SETD6). Negative control (Negative) refers to reaction conducted without addition of E2F1 primary antibody. Number of analyzed GFP positive cells: 19, 18, 28.

Article Snippet: PLA Duolink assays were performed according to the manufacturer’s instructions (Sigma) using antibodies against BRD4 (Bethyl, A700-004), E2F1 (SantaCruz, SC-251) and Flag (Sigma, F1804) overnight at 4°C.

Techniques: Binding Assay, Transfection, Purification, Western Blot, Copurification, Isolation, Control, Microscopy, Histone Deacetylase Assay, Negative Control, Over Expression

Journal: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: E2F1-BRD4 co-occurence is observed in SETD6 KO but not SETD6 WT cells. ( A ) Heatmap of RPKM-normalized E2F1 ChIP-seq signals at E2F1 peaks (±0.8 kb) showing differential chromatin binding of E2F1 in SETD6 WT and KO cells stably expressing Flag-E2F1. The third heatmap shows BRD4 Chip-seq signals in a prostate cancer cell line (SRR1170714) using the same clustering. See also . ( B ) Example browser views showing ChIP-seq of BRD4 (SRR1170714, green) and E2F1 in SETD6 WT and KO cells. See also for additional examples. ( C ) Correlation analysis of E2F1 binding in SETD6 WT and KO cells with the literature BRD4 chromatin binding profile used in panel (A). E2F1 and BRD4 signals were determined in the E2F1 peak regions shown in panel (A) and their correlation was determined. ( D ) Bar-graph showing the slope of the correlation line of BRD4 and E2F1 binding signals in SETD6 WT or KO cells determined using three BRD4 ChIP-seq data sets (datasets SRR1170714, SRR5467129, and SRR5467130). The corresponding analyses are shown in panel (C) and . P -value determined by two-flanked t -test assuming equal variance.

Article Snippet: PLA Duolink assays were performed according to the manufacturer’s instructions (Sigma) using antibodies against BRD4 (Bethyl, A700-004), E2F1 (SantaCruz, SC-251) and Flag (Sigma, F1804) overnight at 4°C.

Techniques: ChIP-sequencing, Binding Assay, Stable Transfection, Expressing

Journal: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: Promoter and enhancer binding of BRD4 at genes preferentially bound by E2F1 and upregulated in SETD6 WT or KO context. ( A ) Average of aggregated BRD4 signals at promoter and enhancer elements of genes preferentially bound by E2F1 and upregulated in SETD6 WT or KO context. Note stronger binding in SETD6 KO cells. P -value determined by two-flanked t -test assuming equal variance. ( B ) Representative genome browser views showing co-occupancy of BRD4 and E2F1 at five genomic regions in SETD6 KO cells: C6orf226, TBCC, RPL21, RPL38, and MYC. ChIP-seq tracks were visualized using IGV (version 2.13.1) displaying BRD4 (SRR1170714, green), E2F1 in SETD6 WT (blue), and E2F1 in SETD6 KO (red) DU145 cells. ( C ) ChIP from SETD6 WT and KO DU145 cells performed using a BRD4-specific antibody to enrich BRD4-bound chromatin fragments. IgG was used as a negative control to assess the specificity of the immunoprecipitation. BRD4 occupancy was evaluated by qPCR at the same loci as shown in panel (B). Two independent biological replicates with three technical repeats were performed. Statistical significance was determined using a two-tailed t -test assuming equal variance. The negative controls RPL21 and RPL28 did not yield a detectable signal. Note the elevated BRD4 binding in SETD6 KO context. ( D ) RT-qPCR analysis of the relative expression of the five target genes shown in panel (B) in untreated SETD6 WT and KO DU145 cells (control) as well as after addition of DMSO and JQ1. Note the strong effect of JQ1 on gene expression in SETD6 KO cells.

Article Snippet: PLA Duolink assays were performed according to the manufacturer’s instructions (Sigma) using antibodies against BRD4 (Bethyl, A700-004), E2F1 (SantaCruz, SC-251) and Flag (Sigma, F1804) overnight at 4°C.

Techniques: Binding Assay, ChIP-sequencing, Negative Control, Immunoprecipitation, Two Tailed Test, Quantitative RT-PCR, Expressing, Control, Gene Expression

Journal: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: Summary of the results of this study. SETD6 monomethylates E2F1 at K117. This methylation disrupts the E2F1–BRD4 interaction leading to different target loci being bound by both factors. In the absence of K117 methylation, E2F1 is acetylated at K117 and K120 leading to BRD4 binding and a concerted engagement of both protein at genomic target sites. As a consequence, methylated and unmethylated E2F1 regulates distinct gene sets in prostate cancer cells.

Article Snippet: PLA Duolink assays were performed according to the manufacturer’s instructions (Sigma) using antibodies against BRD4 (Bethyl, A700-004), E2F1 (SantaCruz, SC-251) and Flag (Sigma, F1804) overnight at 4°C.

Techniques: Methylation, Binding Assay

Journal: Respiratory Research

Article Title: Targeting BRD4 ameliorates experimental emphysema by disrupting super-enhancer in polarized alveolar macrophage

doi: 10.1186/s12931-025-03120-0

Figure Lengend Snippet: BRD4 was associated with key clinical parameters during COPD progression. a Box plots showed the abundance of BRD1 , BRD2 , BRD3 , BRD4 , BRD7 , BRD8 , BRD9 in COPD (n = 94) and control (n = 36). Significance was determined using a two-sided Wilcoxon rank-sum test. NS: Not significant; b Pairwise correlations between BRD1 , BRD2 , BRD3 , BRD4 , BRD7 , BRD8 , BRD9 and clinical parameters of COPD patients. Significant p -values are shown by color, and Spearman’s correlation analysis was used to estimate significant correlations. Clinical parameters included: BMI: body mass index; postFEV1pct: post FEV1 (% reference); pack year: cigarette consumption per year; CAT: COPD Assessment Test; mMRC: modified Medical Research Council; AE_year_0: acute exacerbation times per year; c Correlations of the expression of BRD4 between FEV1% predicted, mMRC and LAA_950 in Guangzhou and Shenzhen COPD cohorts. d Correlations of the expression of between BRD4 and Macrophage ES or Neutrophil ES in the Guangzhou cohort. e BRD4 expression levels in lung tissue were detected by western blotting in control and model mice (n = 3). f Semiquantitative analysis of western blotting results of BRD4 expression levels in the lung. g Immunofluorescence staining of CD68 and BRD4 in lung tissue of control and model mice. Original magnifications 200 × (left) and 630 × (right) (n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Subsequently, the protein was transferred onto a PVDF membrane (Merck Millipore, Bedford, MA, USA) and incubated overnight at 4 °C with antibodies against BRD4 (1:1000; A301985A100, Thermo-fisher, USA) and β-actin (1:3000; AB2001, AB Ways).

Techniques: Control, Modification, Expressing, Western Blot, Immunofluorescence, Staining

Journal: Respiratory Research

Article Title: Targeting BRD4 ameliorates experimental emphysema by disrupting super-enhancer in polarized alveolar macrophage

doi: 10.1186/s12931-025-03120-0

Figure Lengend Snippet: Pharmacological targeting of BRD4 ameliorated LPS/elastase-induced airway inflammation and emphysema. a Measurement of obstructive airflow limitation with FEV100/FVC (%), FRC, and TLC b Measurement of inflammatory cells, macrophages, neutrophils, and lymphocytes in BALF. c Representative H&E-stained pulmonary sections were isolated from different groups. Original magnifications 400 ×. d Semiquantitative analysis of airway inflammation. e Semiquantitative analysis of MLI, n = 5–10 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Subsequently, the protein was transferred onto a PVDF membrane (Merck Millipore, Bedford, MA, USA) and incubated overnight at 4 °C with antibodies against BRD4 (1:1000; A301985A100, Thermo-fisher, USA) and β-actin (1:3000; AB2001, AB Ways).

Techniques: Staining, Isolation

Journal: Respiratory Research

Article Title: Targeting BRD4 ameliorates experimental emphysema by disrupting super-enhancer in polarized alveolar macrophage

doi: 10.1186/s12931-025-03120-0

Figure Lengend Snippet: Transcriptome characterization of BRD4 inhibition in M1 AMs. a Pairwise correlations between BRD1 , BRD2 , BRD3 , BRD4 , BRD7 , BRD8 , BRD9 , and M1 macrophage marker genes expression. b Barplot showed the expression of Tnf , Il1b and IL-6 (n = 5) in the lung of emphysema mice treated with or without DMSO, ARV825 (10 mg/kg), ARV825 (20 mg/kg), JQ1 (50 mg/kg). c Heatmap analysis of RNA-seq data change of M1 macrophage when using 3 different BRD4 inhibition drugs. d Venn diagrams showing overlapped genes between M1-specific genes and genes regulated by 3 different BRD4 inhibition drugs. e GSEA enrichment analysis of ARV-825 regulated genes against M1-specific genes in M1 AMs. f GO analysis of ARV825 downregulated genes in M1 AMs. g Heatmap of gene expression in the pathway of leukocyte migration, cytokine production, and IL-6 production of M1 macrophage. h Barplot showed expression of Tnf, Il1b, and Nos2 (n = 3) in the AMs stimulated with LPS/IFN-γ treated with or without DMSO, ARV825 (0.2 µmol), JQ1 (1 µmol). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Subsequently, the protein was transferred onto a PVDF membrane (Merck Millipore, Bedford, MA, USA) and incubated overnight at 4 °C with antibodies against BRD4 (1:1000; A301985A100, Thermo-fisher, USA) and β-actin (1:3000; AB2001, AB Ways).

Techniques: Inhibition, Marker, Expressing, RNA Sequencing Assay, Migration

Journal: Respiratory Research

Article Title: Targeting BRD4 ameliorates experimental emphysema by disrupting super-enhancer in polarized alveolar macrophage

doi: 10.1186/s12931-025-03120-0

Figure Lengend Snippet: Transcriptome characterization of BRD4 inhibition in M2 AMs. a Pairwise correlations between BRD1 , BRD2 , BRD3 , BRD4 , BRD7 , BRD8 , BRD9 , and M2 macrophage expression in Guangzhou and Shenzhen clinical cohorts. b Barplot showed expression of Arg1 , Ccl17 , and Tfrc (n = 5) in the lung of emphysema mice treated with or without DMSO, ARV825 (10 mg/kg), ARV825 (20 mg/kg), and JQ1 (50 mg/kg). c Heatmap of RNA-seq data of M2 macrophage when using 3 different BRD4 inhibition drugs. d Venn diagrams showing overlapped genes between M2-specific genes and genes downregulated using 3 different BRD4 inhibition drugs. e GSEA enrichment analysis of ARV825 regulated genes against M2-specific gene list. f GO analysis of the differentially expressed mRNAs in BRD4 inhibition drugs of ARV825 downregulated genes. (G) Heatmap analysis of gene expression change in myeloid leukocyte differentiation, leukocyte chemotaxis, and extracellular matrix organization of M2 macrophage when using 3 different BRD4 inhibition drugs. h Barplot showed expression of Arg1 , Ccl17 (n = 3) in the AMs stimulated with IL-4 in the treatment with or without DMSO, ARV825 (0.2 µmol), JQ1 (1 µmol). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Subsequently, the protein was transferred onto a PVDF membrane (Merck Millipore, Bedford, MA, USA) and incubated overnight at 4 °C with antibodies against BRD4 (1:1000; A301985A100, Thermo-fisher, USA) and β-actin (1:3000; AB2001, AB Ways).

Techniques: Inhibition, Expressing, RNA Sequencing Assay, Chemotaxis Assay

Journal: Respiratory Research

Article Title: Targeting BRD4 ameliorates experimental emphysema by disrupting super-enhancer in polarized alveolar macrophage

doi: 10.1186/s12931-025-03120-0

Figure Lengend Snippet: BRD4 inhibition decreased BRD4 and H3K27ac occupancy on promoter regions in vitro. a Venn diagram showing BRD4 and H3K27ac overlapped binding peaks in M0 and M2 AMs. b Genome-wide distribution of BRD4 and H3k27ac in AMs from M0 group, M2 group, ARV-825 + M2 group. c Heatmaps of BRD4 and H3k27ac occupancy on the promoter region (TSS ± 5 kb), aligned by the degree of BRD4 and H3k27ac signal intensity in AMs from M0 group, M2 group, ARV-825 + M2 group. d Binding intensity of BRD4 and H3k27ac across the gene body of the whole genome in AMs from M0 group, M2 group, ARV-825 + M2 group

Article Snippet: Subsequently, the protein was transferred onto a PVDF membrane (Merck Millipore, Bedford, MA, USA) and incubated overnight at 4 °C with antibodies against BRD4 (1:1000; A301985A100, Thermo-fisher, USA) and β-actin (1:3000; AB2001, AB Ways).

Techniques: Inhibition, In Vitro, Binding Assay, Genome Wide

Journal: Respiratory Research

Article Title: Targeting BRD4 ameliorates experimental emphysema by disrupting super-enhancer in polarized alveolar macrophage

doi: 10.1186/s12931-025-03120-0

Figure Lengend Snippet: BRD4 inhibition changed the dynamic enhancer epigenome in M2 AMs. a The left Venn diagram showed enriched genes annotated to BRD4 peaks, H3K27ac peaks in M2 AMs, and their overlapped genes with upregulated genes in M2 macrophage; the right Venn diagram showed overlapped genes between genes associated with ARV825 downregulated BRD4 peaks, genes associated with ARV825 downregulated H3K27ac peaks, and ARV825 downregulated genes in M2 AMs. b The boxplot showed significantly differential gene expression fold changes between genes associated with and not with downregulated BRD4 peaks and H3K27ac peaks in M2 AMs with ARV-825. c GSEA plots indicated genes associated with downregulated BRD4 and H3k27ac peaks are significantly downregulated by ARV-825 in M2 macrophage. d GO analysis of the ARV-825 downregulated genes associated with BRD4 and H3k27ac downregulated peaks in M2 AMs. e Gene tracks showed strong downregulated signals of the M2 marker genes Arg1 , Ccl17 , Ccr5 , and Ccl22 after ARV-825 treatment. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Subsequently, the protein was transferred onto a PVDF membrane (Merck Millipore, Bedford, MA, USA) and incubated overnight at 4 °C with antibodies against BRD4 (1:1000; A301985A100, Thermo-fisher, USA) and β-actin (1:3000; AB2001, AB Ways).

Techniques: Inhibition, Expressing, Marker

Journal: Respiratory Research

Article Title: Targeting BRD4 ameliorates experimental emphysema by disrupting super-enhancer in polarized alveolar macrophage

doi: 10.1186/s12931-025-03120-0

Figure Lengend Snippet: BRD4 inhibition disrupted IRF4 SE formation in M2 AMs. a Super enhancers of M0, M2, and ARV825-M2 macrophage, which are ranked by their H3K27ac signaling using ROSE. b Venn diagram showed the overlap of genes associated with SEs sites, M2-specific genes, and ARV825 downregulated genes in M2. c Gene tracks of Irf4 associated SE in M2 AMs. The adjacent genes IRF4 showed strong SE peaks and RNA-seq signals in M2 AMs and intensively reduced when using ARV-825. d Volcano plot depicting significant downregulation of Irf4 in M2 macrophage by BRD4 inhibition drug ARV825. e Venn diagram showed overlapped peaks of BRD4, H3K27ac, and IRF4 in M2 AMs. f Binding intensity of IRF4 across the BRD4 binding sites of the whole genome in AMs from the M0 group, M2 group, and ARV-825 + M2 group. g Gene tracks of Mmp12 in the M2 AMs. The adjacent gene Mmp12 showed strong IRF4 peaks and RNA-seq signals in the M2 AMs. h Barplot showed expression of Irf4 , Mmp12 , and Tfrc in response to IRF4 transfection in M2 AMs with or without treatment with ARV825 (n = 5). i Transcription level of Irf4, Mmp12 was significantly downregulated by BRD4 inhibition drugs in pulmonary sections from LPS/elastase-induced COPD mice. The values were detected by quantitative PCR (n = 5). j Box plots showed the abundance of IRF4 (Guangzhou p = 0.006; Shenzhen p = 0.0012), and MMP12 (Guangzhou p = 0.00056; Shenzhen p = 0.00022) in COPD and healthy control. k The gene expression of IRF4 and MMP12 were significantly correlated in Guangzhou and Shenzhen COPD patients. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Subsequently, the protein was transferred onto a PVDF membrane (Merck Millipore, Bedford, MA, USA) and incubated overnight at 4 °C with antibodies against BRD4 (1:1000; A301985A100, Thermo-fisher, USA) and β-actin (1:3000; AB2001, AB Ways).

Techniques: Inhibition, RNA Sequencing Assay, Binding Assay, Expressing, Transfection, Real-time Polymerase Chain Reaction, Control