Journal: Molecular Cell

Article Title: Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function

doi: 10.1016/j.molcel.2017.04.027

Figure Lengend Snippet: BRD4 Silencing Enhances Autophagic Flux (A) Drosophila S2R + cells expressing GFP-LC3 were transfected with double-stranded RNA (dsRNA) targeting control luciferase (Luc) or Fs(1)h. (B and C) KP-4 cells transfected with control or BRD4 siRNA for 72 hr were subjected to western blot analysis (B) and stained for LC3B (C). The number of LC3 puncta normalized to cell number is shown. CON: n = 94 cells, BRD4 1: n = 97 cells, BRD4 2: n = 74 cells. Scale bars, 50 μm. (D) Immunohistochemistry of small intestinal sections from transgenic mice harboring inducible renilla luciferase or BRD4 shRNA. Sections were stained for LC3 (upper) and BRD4 (lower). Cytoplasmic signal in BRD4 panels is due to non-specific staining. Scale bars, 50 μm. (E) KP-4 cells transfected with BRD4 siRNA were treated with 10 μM CQ for 4 hr. (F) KP-4 cells transfected with BRD4 siRNA were stained for WIPI2. The number of WIPI2 puncta normalized to cell number is shown. CON: n = 119 cells, BRD4 1: n = 107 cells, BRD4 2: n = 109 cells. Scale bars, 20 μm. (G) KP-4 cells stably expressing RFP-GFP-LC3 were transfected with BRD4 siRNA. Scale bars, 50 μm. (H) KP-4 cells were treated with 500 nM JQ1 for 9 hr in the presence or absence of CQ (10 μM, 4 hr). (I) KP-4 cells overexpressing BRD4 were treated with 10 μM CQ for 4 hr. (J) TY-82 cells transfected with NUT siRNA for 5 days were treated with 10 μM CQ for 8 hr. BRD4-NUT was detected using NUT antibody. All data are shown as mean ± SD. ∗ p < 0.01. See also Figure S1 .

Article Snippet: The sections were incubated with LC3B (NanoTools, Cat#: 0231-100/LC3-5F10, 1/50) or BRD4 (Bethyl Laboratories, Cat#: A301-985A50, 1/2000) antibody diluted in Antibody diluent (Dako, Cat#: S2022) for 35 min. After wash with TBST twice, the sections were incubated with EnVision+ HRP, Mouse or Rabbit (Dako, Cat#: K4001 and K4003) for 30 min followed by wash with TBST twice.

Techniques: Expressing, Transfection, Control, Luciferase, Western Blot, Staining, Immunohistochemistry, Transgenic Assay, shRNA, Stable Transfection

Journal: Molecular Cell

Article Title: Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function

doi: 10.1016/j.molcel.2017.04.027

Figure Lengend Snippet: BRD4 Is a Negative Regulator of Autophagy Gene Expression (A and B) KP-4 cells transfected with control or BRD4 siRNA were subjected to RNA-seq and gene ontology analyses (A) and RT-qPCR analysis (B). (C) RT-qPCR analysis of KP-4 cells treated with DMSO, 500 nM JQ1, 500 nM I-BET151, or 500 nM OTX015 for 9 hr. (D) RT-qPCR analysis of KP-4 cells treated with 500 nM JQ1 for the indicated time. (E) RT-qPCR analysis of KP-4 cells overexpressing BRD4. All data are shown as mean ± SD. In (A)–(D), n = 3 independent experiments; in (E), data are representative of two independent experiments performed in triplicate. ∗ p < 0.01, ∗∗ p < 0.05. See also Figure S2 .

Article Snippet: The sections were incubated with LC3B (NanoTools, Cat#: 0231-100/LC3-5F10, 1/50) or BRD4 (Bethyl Laboratories, Cat#: A301-985A50, 1/2000) antibody diluted in Antibody diluent (Dako, Cat#: S2022) for 35 min. After wash with TBST twice, the sections were incubated with EnVision+ HRP, Mouse or Rabbit (Dako, Cat#: K4001 and K4003) for 30 min followed by wash with TBST twice.

Techniques: Gene Expression, Transfection, Control, RNA Sequencing, Quantitative RT-PCR

Journal: Molecular Cell

Article Title: Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function

doi: 10.1016/j.molcel.2017.04.027

Figure Lengend Snippet: BRD4 Knockdown Enhances Lysosomal Function (A) RT-qPCR analysis of KP-4 cells transfected with control or BRD4 siRNA. (B–D) KP-4 cells transfected with BRD4 siRNA were subjected to western blot analysis with antibodies against lysosomal proteins (B) and stained with LAMP1 antibody (C), LysoTracker Red (100 nM, 2 hr) (D, upper panels), and Magic Red CTSB (1 hr) (D, lower panels). Area of LAMP1 + , LysoTracker + , and Magic Red CTSB + area normalized to cell number is shown (C, CON: n = 115 cells, BRD4: n = 130 cells; D upper, CON: n = 66 cells, BRD4 1: n = 52 cells, BRD4 2: n = 50 cells; D lower, CON: n = 164 cells, BRD4 1: n = 109 cells, BRD4 2: n = 53 cells). Scale bars, 50 μm. (E) Hexosaminidase activity was measured using lysates from control and BRD4 knockdown KP-4 cells. (F and G) RT-qPCR analysis of TY-82 cells transfected with NUT siRNA for 72 hr (F) or treated with 500 nM JQ1 for 9 hr (G). (H) KP-4 cells were transfected with BRD4 and/or MiT/TFE (TFEB, TFE3, MITF) siRNAs and treated with 10 μM CQ for 4 hr. All data are shown as mean ± SD. In (A) and (F), n = 3 independent experiments. In (E), n = 4 independent experiments. In (G), data are representative of two independent experiments performed in triplicate. ∗ p < 0.01, ∗∗ p < 0.05. See also Figure S3 .

Article Snippet: The sections were incubated with LC3B (NanoTools, Cat#: 0231-100/LC3-5F10, 1/50) or BRD4 (Bethyl Laboratories, Cat#: A301-985A50, 1/2000) antibody diluted in Antibody diluent (Dako, Cat#: S2022) for 35 min. After wash with TBST twice, the sections were incubated with EnVision+ HRP, Mouse or Rabbit (Dako, Cat#: K4001 and K4003) for 30 min followed by wash with TBST twice.

Techniques: Knockdown, Quantitative RT-PCR, Transfection, Control, Western Blot, Staining, Activity Assay

Journal: Molecular Cell

Article Title: Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function

doi: 10.1016/j.molcel.2017.04.027

Figure Lengend Snippet: Starvation Leads to BRD4 Dissociation from Autophagy Gene Promoters (A and B) KP-4 cells were starved for 4 hr followed by chromatin immunoprecipitation (ChIP) assay with BRD4 antibody (A) and RT-qPCR analysis (B). (C) KP-4 cells infected with Cas9/hMOF sgRNA were subjected to ChIP assay with BRD4 antibody. (D) KP-4 cells were starved for 4 hr followed by ChIP assay with H4K16Ac antibody. (E and F) KP-4 cells infected with Cas9/SIRT1 sgRNA were starved for 4 hr followed by ChIP assay with BRD4 antibody (E) and RT-qPCR analysis (F). Western blot shows efficient SIRT1 depletion in Cas9/SIRT1 sgRNA-infected cells. (G and H) KP-4 cells infected with Cas9/AMPKα1 and α2 sgRNAs were starved for 4 hr followed by immunoprecipitation with DBC1 antibody (G) and RT-qPCR analysis (H). All data are shown as mean ± SD. In (A)–(F) and (H), n = 3 independent experiments. ∗ p < 0.01, ∗∗ p < 0.05, N.S., no significance. See also Figure S4 .

Article Snippet: The sections were incubated with LC3B (NanoTools, Cat#: 0231-100/LC3-5F10, 1/50) or BRD4 (Bethyl Laboratories, Cat#: A301-985A50, 1/2000) antibody diluted in Antibody diluent (Dako, Cat#: S2022) for 35 min. After wash with TBST twice, the sections were incubated with EnVision+ HRP, Mouse or Rabbit (Dako, Cat#: K4001 and K4003) for 30 min followed by wash with TBST twice.

Techniques: Chromatin Immunoprecipitation, Quantitative RT-PCR, Infection, Western Blot, Immunoprecipitation

Journal: Molecular Cell

Article Title: Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function

doi: 10.1016/j.molcel.2017.04.027

Figure Lengend Snippet: BRD4 Represses Autophagy Gene Expression through G9a (A) Cell extracts from KP-4 cells were subjected to immunoprecipitation with G9a (upper) and BRD4 (lower) antibodies. (B) KP-4 cells were starved for 4 hr followed by immunoprecipitation with BRD4 antibody. (C–F) KP-4 cells were treated with 500 nM JQ1 for 9 hr (C and E). KP-4 cells harboring inducible control or BRD4 shRNA were treated with 500 ng/mL doxycycline (DOX) for 4 days (D and F). ChIP assays were performed using G9a (C and D) and H3K9diMe (E and F) antibodies. (G and H) KP-4 cells infected with shRNA targeting G9a were transfected with BRD4 siRNA followed by RT-qPCR (G) and western blot (H). (I) KP-4 cells overexpressing BRD4 were infected with shRNA targeting G9a. All data are shown as mean ± SD. In (C)–(G), n = 3 independent experiments. ∗ p < 0.01, ∗∗ p < 0.05, N.S., no significance. See also Figure S5 .

Article Snippet: The sections were incubated with LC3B (NanoTools, Cat#: 0231-100/LC3-5F10, 1/50) or BRD4 (Bethyl Laboratories, Cat#: A301-985A50, 1/2000) antibody diluted in Antibody diluent (Dako, Cat#: S2022) for 35 min. After wash with TBST twice, the sections were incubated with EnVision+ HRP, Mouse or Rabbit (Dako, Cat#: K4001 and K4003) for 30 min followed by wash with TBST twice.

Techniques: Gene Expression, Immunoprecipitation, Control, shRNA, Infection, Transfection, Quantitative RT-PCR, Western Blot

Journal: Molecular Cell

Article Title: Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function

doi: 10.1016/j.molcel.2017.04.027

Figure Lengend Snippet: Effect of BRD4 Silencing on Stimulus-Dependent and Selective Autophagy (A–F) Cells transfected with BRD4 siRNA were starved for 1–5 hr (KP-4 cells, A), treated with 500 nM rapamycin for 24 hr (KP-4 cells, B), starved of glucose for 4 hr (KP-4 cells, C), cultured under hypoxic (1% O 2 ) conditions for 48 hr (SUIT2 cells, D), treated with 100 mM Trehalose for 4 hr (KP-4 cells, E), or treated with 500 nM 4-Hydroxytamoxifen (4-OHT) for 48 hr (IMR90 ER-HRas G12V cells, F). (G) KP-4 cells harboring rtTA and Tre-tight-HTT Q94-CFP were transfected with BRD4 siRNA. At 12 hr after transfection, cells were treated with 1 μg/mL DOX for 10 hr. At 48 hr after removal of DOX, cells were separated into Triton X-100 soluble and insoluble fractions. (H) KP-4 cells transfected with BRD4 siRNA were infected with Salmonella enterica serovar Typhimurium. The number of Salmonella was determined by performing colony-forming unit assays at 2, 6, and 8 hr after infection and normalized to the numbers at 2 hr. Data are shown as mean ± SEM; n = 4 independent experiments. (I) KP-4 cells expressing YFP-parkin were transfected with BRD4 siRNA followed by treatment with 1 μM Antimycin A and 1 μM Oligomycin for 8 hr. See also Figure S6 .

Article Snippet: The sections were incubated with LC3B (NanoTools, Cat#: 0231-100/LC3-5F10, 1/50) or BRD4 (Bethyl Laboratories, Cat#: A301-985A50, 1/2000) antibody diluted in Antibody diluent (Dako, Cat#: S2022) for 35 min. After wash with TBST twice, the sections were incubated with EnVision+ HRP, Mouse or Rabbit (Dako, Cat#: K4001 and K4003) for 30 min followed by wash with TBST twice.

Techniques: Transfection, Cell Culture, Infection, Expressing

Journal: Molecular Cell

Article Title: Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function

doi: 10.1016/j.molcel.2017.04.027

Figure Lengend Snippet: BRD4 Knockdown Sustains mTOR Activity during Starvation and Confers Resistance to Starvation-Induced Cell Death (A and B) KP-4 cells transfected with BRD4 siRNA were starved of amino acids (A). Cells pre-treated with CQ (10 μM, 4 hr) were subjected to amino acid starvation for 2 hr in the presence of CQ (B). (C) KP-4 cells infected with Cas9/ATG5 sgRNA were transfected with BRD4 siRNA and subjected to amino acid starvation for 2 hr. (D and E) KP-4 cells infected with Cas9/ATG5 sgRNA were transfected with BRD4 siRNA. Following 48 hr starvation, percentage of subG1 cells (D) and cell number (E) were determined (n = 3 independent experiments). (F and G) KP-4 cells transfected with BRD4 siRNA were starved for 48 hr in the presence or absence of 10 μM CQ. Percentage of dead (F) and surviving (G) cells was determined by trypan blue exclusion test (n = 4 independent experiments). All data are shown as mean ± SD. ∗ p < 0.01, N.S., no significance. See also Figure S7 .

Article Snippet: The sections were incubated with LC3B (NanoTools, Cat#: 0231-100/LC3-5F10, 1/50) or BRD4 (Bethyl Laboratories, Cat#: A301-985A50, 1/2000) antibody diluted in Antibody diluent (Dako, Cat#: S2022) for 35 min. After wash with TBST twice, the sections were incubated with EnVision+ HRP, Mouse or Rabbit (Dako, Cat#: K4001 and K4003) for 30 min followed by wash with TBST twice.

Techniques: Knockdown, Activity Assay, Transfection, Infection

Journal: Molecular Cell

Article Title: Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function

doi: 10.1016/j.molcel.2017.04.027

Figure Lengend Snippet:

Article Snippet: The sections were incubated with LC3B (NanoTools, Cat#: 0231-100/LC3-5F10, 1/50) or BRD4 (Bethyl Laboratories, Cat#: A301-985A50, 1/2000) antibody diluted in Antibody diluent (Dako, Cat#: S2022) for 35 min. After wash with TBST twice, the sections were incubated with EnVision+ HRP, Mouse or Rabbit (Dako, Cat#: K4001 and K4003) for 30 min followed by wash with TBST twice.

Techniques: Control, Membrane, Virus, Recombinant, Sample Prep, Flow Cytometry, Western Blot, Microscopy, Plasmid Preparation, Phospho-proteomics, Variant Assay, shRNA, Software, CRISPR

Journal: Antiviral research

Article Title: Screening of an epigenetic compound library identifies BRD4 as a potential antiviral target for hepatitis B virus covalently closed circular DNA transcription.

doi: 10.1016/j.antiviral.2023.105552

Figure Lengend Snippet: Fig. 8. MS436-mediated epigenetic change of cccDNA minichromosome. (A–D) HepAD38 cells cultured in T75 flask were induced in Tet-free me dium for 10 days for cccDNA establishment, then treated with DMSO or MS436 (10 μM) every other day for 4 days in the presence of 3TC (10 μM). Cells were crosslinked and lysed for further chromatin fragmentation by sonication-based shearing. cccDNA was immunoprecipitated by ChIP-grade non-immune IgG isotype control or antibodies against H3K27ac, H3K9me3, H3K4me3 or BRD4 and detected by qPCR. The enrichment of aforementioned histone PTMs or BRD4 protein on cccDNA was plotted as fold change to NIS IgG control, respectively. Data are shown as mean ± SD (n = 3). **p < 0.01, ***p < 0.001, ns: not significant. (E) The intracellular total BRD4 proteins, including both the long and short isoforms, were detected by western blot. β-actin served as the loading control.

Article Snippet: BRD4 degrader MZ1 (cat# S8889) and HBV replication inhibitor lamivudine (3TC) (cat# S1706) were purchased from Selleck Chemicals.

Techniques: Cell Culture, Sonication, Immunoprecipitation, Control, Western Blot

Journal: Antiviral research

Article Title: Screening of an epigenetic compound library identifies BRD4 as a potential antiviral target for hepatitis B virus covalently closed circular DNA transcription.

doi: 10.1016/j.antiviral.2023.105552

Figure Lengend Snippet: Fig. 11. siRNA knock down of BRD4 reduces HBV transcription. HepG2-NTCP cells in 6-well-plate were transfected with control or BRD4 siRNA for 2 days, followed by HBV infection with 500 vge/cell for 24 h, then a second round of siRNA transfection was con ducted in the virally infected cells. The cells were cultured for another 2 days, (A) cellular BRD4 and β-actin proteins were detected by western blot; the intracellular HBV RNA and cccDNA were analyzed by northern blot and qPCR, respectively. (B) The relative cccDNA levels are plotted as fold change to siControl. The relative levels of HBV mRNA normalized to cccDNA are plotted as fold change to that of siCon trol. Data are shown as mean ± SD, n = 3; ***p < 0.001.

Article Snippet: BRD4 degrader MZ1 (cat# S8889) and HBV replication inhibitor lamivudine (3TC) (cat# S1706) were purchased from Selleck Chemicals.

Techniques: Knockdown, Transfection, Control, Infection, Cell Culture, Western Blot, Northern Blot

Journal: Antiviral research

Article Title: Screening of an epigenetic compound library identifies BRD4 as a potential antiviral target for hepatitis B virus covalently closed circular DNA transcription.

doi: 10.1016/j.antiviral.2023.105552

Figure Lengend Snippet: Fig. 12. Depletion of BRD4 by PROTAC degraders inhibits HBV transcription. (A, C) Chemical structure of dBET1 and MZ-1. (B, D) HepG2-NTCP cell were infected with HBV (500 vge/cell) for 6 days, followed by treatment with (B) dBET1 (10 μM) in the absence or presence of 3TC (10 μM) or (D) MZ-1 (1 μM) and 3TC (10 μM) every other day for 4 days. Cellular BRD4 was detected by western blot, β-actin served as loading control. HBV total RNA and/or cytoplasmic core DNA were detected by northern and Southern blot, respectively.

Article Snippet: BRD4 degrader MZ1 (cat# S8889) and HBV replication inhibitor lamivudine (3TC) (cat# S1706) were purchased from Selleck Chemicals.

Techniques: Infection, Western Blot, Control, Northern Blot, Southern Blot

Journal: Acta Pharmaceutica Sinica. B

Article Title: Development of hedgehog pathway inhibitors by epigenetically targeting GLI through BET bromodomain for the treatment of medulloblastoma

doi: 10.1016/j.apsb.2020.07.007

Figure Lengend Snippet: The hedgehog signaling pathway and representative inhibitors of SMO, GLI and BRD4. Ac, acetylation; BRD4, bromodomain-containing protein 4; GLI, glioma-associated oncogene homolog protein; GLI A , activated GLI; HH, hedgehog; PTCH, patched; SMO, smoothened; SUFU, suppressor of fused.

Article Snippet: Recombinant BRD4 proteins (Active Motif, Cat#31380 and 31446, Carlsbad, CA, USA), compounds and peptide were diluted to desired working concentrations with EPIgenerous binding buffer (Cisbio, Cat#62DLBDDF, Codolet, France).

Techniques:

Journal: Acta Pharmaceutica Sinica. B

Article Title: Development of hedgehog pathway inhibitors by epigenetically targeting GLI through BET bromodomain for the treatment of medulloblastoma

doi: 10.1016/j.apsb.2020.07.007

Figure Lengend Snippet: Inhibition of clinical BRD4 inhibitors against BRD4 BD1 and the HH pathway a .

Article Snippet: Recombinant BRD4 proteins (Active Motif, Cat#31380 and 31446, Carlsbad, CA, USA), compounds and peptide were diluted to desired working concentrations with EPIgenerous binding buffer (Cisbio, Cat#62DLBDDF, Codolet, France).

Techniques: Inhibition

Journal: Acta Pharmaceutica Sinica. B

Article Title: Development of hedgehog pathway inhibitors by epigenetically targeting GLI through BET bromodomain for the treatment of medulloblastoma

doi: 10.1016/j.apsb.2020.07.007

Figure Lengend Snippet: Inhibition of compounds against BRD4 (BD1) and the HH pathway a .

Article Snippet: Recombinant BRD4 proteins (Active Motif, Cat#31380 and 31446, Carlsbad, CA, USA), compounds and peptide were diluted to desired working concentrations with EPIgenerous binding buffer (Cisbio, Cat#62DLBDDF, Codolet, France).

Techniques: Inhibition

Journal: Acta Pharmaceutica Sinica. B

Article Title: Development of hedgehog pathway inhibitors by epigenetically targeting GLI through BET bromodomain for the treatment of medulloblastoma

doi: 10.1016/j.apsb.2020.07.007

Figure Lengend Snippet: Inhibition of compounds against BRD4 (BD1) and the HH pathway a .

Article Snippet: Recombinant BRD4 proteins (Active Motif, Cat#31380 and 31446, Carlsbad, CA, USA), compounds and peptide were diluted to desired working concentrations with EPIgenerous binding buffer (Cisbio, Cat#62DLBDDF, Codolet, France).

Techniques: Inhibition

Journal: Acta Pharmaceutica Sinica. B

Article Title: Development of hedgehog pathway inhibitors by epigenetically targeting GLI through BET bromodomain for the treatment of medulloblastoma

doi: 10.1016/j.apsb.2020.07.007

Figure Lengend Snippet: hERG inhibition and predicted physico-chemical parameters of representative compounds a .

Article Snippet: Recombinant BRD4 proteins (Active Motif, Cat#31380 and 31446, Carlsbad, CA, USA), compounds and peptide were diluted to desired working concentrations with EPIgenerous binding buffer (Cisbio, Cat#62DLBDDF, Codolet, France).

Techniques: Inhibition

Journal: Acta Pharmaceutica Sinica. B

Article Title: Development of hedgehog pathway inhibitors by epigenetically targeting GLI through BET bromodomain for the treatment of medulloblastoma

doi: 10.1016/j.apsb.2020.07.007

Figure Lengend Snippet: Inhibition of compounds against BRD4 (BD1) and the HH pathway a .

Article Snippet: Recombinant BRD4 proteins (Active Motif, Cat#31380 and 31446, Carlsbad, CA, USA), compounds and peptide were diluted to desired working concentrations with EPIgenerous binding buffer (Cisbio, Cat#62DLBDDF, Codolet, France).

Techniques: Inhibition

Journal: Acta Pharmaceutica Sinica. B

Article Title: Development of hedgehog pathway inhibitors by epigenetically targeting GLI through BET bromodomain for the treatment of medulloblastoma

doi: 10.1016/j.apsb.2020.07.007

Figure Lengend Snippet: Inhibition of compounds against BRD4 (BD1) and the HH pathway a .

Article Snippet: Recombinant BRD4 proteins (Active Motif, Cat#31380 and 31446, Carlsbad, CA, USA), compounds and peptide were diluted to desired working concentrations with EPIgenerous binding buffer (Cisbio, Cat#62DLBDDF, Codolet, France).

Techniques: Inhibition

Journal: Acta Pharmaceutica Sinica. B

Article Title: Development of hedgehog pathway inhibitors by epigenetically targeting GLI through BET bromodomain for the treatment of medulloblastoma

doi: 10.1016/j.apsb.2020.07.007

Figure Lengend Snippet: Inhibition of compounds against BRD4 (BD1) and the HH pathway a .

Article Snippet: Recombinant BRD4 proteins (Active Motif, Cat#31380 and 31446, Carlsbad, CA, USA), compounds and peptide were diluted to desired working concentrations with EPIgenerous binding buffer (Cisbio, Cat#62DLBDDF, Codolet, France).

Techniques: Inhibition

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: Cancer research

Article Title: MDM2-recruiting PROTAC Offers Superior, Synergistic Anti-proliferative Activity via Simultaneous Degradation of BRD4 and Stabilization of p53

doi: 10.1158/0008-5472.CAN-18-2918

Figure Lengend Snippet: (A) structure of the BRD4/BET inhibitor, JQ1; (B) structure of idasanutlin (RG7388), an MDM2 antagonist; (C) structure of A1874, an MDM2-recruiting, BRD4-degrading PROTAC.

Article Snippet: Reagents Antibodies against BRD4 (cat. no. 13440), GAPDH (cat. no. 2118) and p21 CIP1/WAF1 (cat. no. 2947) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques:

Journal: Cancer research

Article Title: MDM2-recruiting PROTAC Offers Superior, Synergistic Anti-proliferative Activity via Simultaneous Degradation of BRD4 and Stabilization of p53

doi: 10.1158/0008-5472.CAN-18-2918

Figure Lengend Snippet: (A) Structure of A1875, a diastereomeric analog of A1874 with greatly reduced affinity for MDM2. (B) Representative immunoblots from HCT116 cells treated with increasing concentrations of A1875. (C) Quantified results of immunoblots in panel (B). (D) Structure of A743, a VHL-recruiting BRD4-degrading PROTAC. (E) Representative immunoblots from HCT116 cells treated with increasing concentrations of A743. (F) Quantified results of immunoblots in panel (E).

Article Snippet: Reagents Antibodies against BRD4 (cat. no. 13440), GAPDH (cat. no. 2118) and p21 CIP1/WAF1 (cat. no. 2947) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot

Journal: Cell Genomics

Article Title: Recurrent breakpoints in the BRD4 locus reduce toxicity associated with gene amplification

doi: 10.1016/j.xgen.2025.100815

Figure Lengend Snippet: Significantly recurrent breakpoints result in focal deletion of BRD4 regulatory regions (A) Top: breakpoint densities for PCAWG breast (BRCA), endometrial (EN) and ovarian (OV) cancers exhibiting rearrangements at the BRD4 locus, compared to an independent breast cancer dataset (Nik-Zainal et al. ); Center: somatic copy-number alterations (SCNAs) for pooled breast, ovarian, and endometrial cancer samples with BRD4 focal deletions. Bottom: alignment with H3K4me3 from breast (MCF-7) and ovarian (HMEC) cell lines. Genomic track: gene exons and intron as defined in RefSeq for hg19. (B and C) (B) Total copy number at CCNE1 locus (chromosome 19) between tumors with or without BRD4 focal deletions and (C) only breast, ovarian, and endometrial tumors; Wilcoxon rank-sum test. The box spans the interquartile range (IQR) with the median as a horizontal line and bars extending to 1.5 × IQR. (D) Proportion of tumors with or without amplifications spanning the BRD4 locus, comparing those with or without a concomitant BRD4 focal deletion.

Article Snippet: After blocking with 5% dry milk in TBS-T for 30 min, the membrane was incubated at 4°C overnight with primary antibodies against rabbit BRD4 (cat. no. 702448, ThermoFisher Scientific), rabbit BRD4-L (cat. no. A301-985A100, Bethyl Laboratories), mouse SpCas9 (cat. no. 14697, Cell Signaling Technology), or rabbit GAPDH (cat. No. 2118, Cell Signaling Technology).

Techniques:

Journal: Cell Genomics

Article Title: Recurrent breakpoints in the BRD4 locus reduce toxicity associated with gene amplification

doi: 10.1016/j.xgen.2025.100815

Figure Lengend Snippet: Focal deletions of BRD4 significantly decrease BRD4 expression across isoforms (A) Volcano plot of global gene expression comparing BRD4 focal deletion tumors ( n = 22) with all other PCAWG tumors ( n = 2,636), Bonferroni-corrected cutoff of p < 0.1. Genes on chromosome 19 are displayed in bold type. (B) Expression of BRD4-L (ENST00000263377; BRD4-201) and BRD4-S(a) (ENST00000371835; BRD-203) isoforms in normal tissues as obtained from GTEx (TPM, transcripts per million). Two-tailed Wilcoxon signed rank test with Bonferroni-corrected p values as indicated. (C and D) Isoform-specific expression of BRD4 in PCAWG breast cancer (C) and ovarian (D) tumors, normalized to copy number, comparing BRD4-L (ENST00000263377; BRD4-201) with two BRD4-S isoforms (dominant BRD4-S(a): ENST00000371835, BRD-203, and secondary BRD4-S(b): ENST00000360016, BRD4-202) between tumors with or without BRD4 focal deletions (FPKM, fragments per kilobase of transcript per million fragments mapped). Wilcoxon rank-sum test. The box spans the IQR with the median as a horontal line and error bars extending to 1.5. (E) Coefficients of a linear model for BRD4 expression controlling for tumor type (non-breast, ovarian, or endometrial as baseline), absolute copy number, and focal deletion status (not deleted as baseline). Error bars represent the 95% confidence intervals for coefficient estimates from the linear model.

Article Snippet: After blocking with 5% dry milk in TBS-T for 30 min, the membrane was incubated at 4°C overnight with primary antibodies against rabbit BRD4 (cat. no. 702448, ThermoFisher Scientific), rabbit BRD4-L (cat. no. A301-985A100, Bethyl Laboratories), mouse SpCas9 (cat. no. 14697, Cell Signaling Technology), or rabbit GAPDH (cat. No. 2118, Cell Signaling Technology).

Techniques: Expressing, Gene Expression, Two Tailed Test

Journal: Cell Genomics

Article Title: Recurrent breakpoints in the BRD4 locus reduce toxicity associated with gene amplification

doi: 10.1016/j.xgen.2025.100815

Figure Lengend Snippet: BRD4 overexpression is toxic across multiple cancer types (A) Overview of an ORF screen, from which the effect of BRD4-L and BRD4-S(a) overexpression on cell proliferation can be assessed. (B) Log 2 -fold changes in cell proliferation for BRD4 constructs, expressed relative to the experimental early time point (ETP) of each screen. Negative changes reflect a detrimental effect on cell proliferation. ORF screens were conducted in breast (BRCA), ovarian (OV), neuroblastoma (NB), skin cutaneous melanoma (SKCM), lung adenocarcinoma (LUAD), Ewing sarcoma (EWS), medulloblastoma (MB), and prostate adenocarcinoma (PRAD) cell lines. (C) ORF screen performance between BRD4-L and BRD4-S(a) isoforms for each cell line. Two-tailed t test. (D) Validation of BRD4 isoform expression in OVSAHO cells. Transcript expression is quantified relative to GFP control. Mean and standard deviation of three replicates; two-way ANOVA. (E) Immunoblot of BRD4-L and BRD4-S protein variants detected in OVSAHO cells transduced with BRD4 or control GFP overexpression constructs. Values represent levels of protein expression, normalized to glyceraldehyde 3-phosphate dehydrogenase expression and shown relative to parental cells (endogenous BRD4 levels). (F) Quantification of cell confluency in OVSAHO-Cas9 after transduction with BRD4-L, BRD4-S(a), or GFP control overexpressing constructs. Mean and standard deviation of three replicates, two-way ANOVA followed by Dunnett post-tests, controlling the Family-wise alpha threshold and confidence level. p values of the final time point are annotated.

Article Snippet: After blocking with 5% dry milk in TBS-T for 30 min, the membrane was incubated at 4°C overnight with primary antibodies against rabbit BRD4 (cat. no. 702448, ThermoFisher Scientific), rabbit BRD4-L (cat. no. A301-985A100, Bethyl Laboratories), mouse SpCas9 (cat. no. 14697, Cell Signaling Technology), or rabbit GAPDH (cat. No. 2118, Cell Signaling Technology).

Techniques: Over Expression, Construct, Two Tailed Test, Biomarker Discovery, Expressing, Control, Standard Deviation, Western Blot, Transduction

Journal: Cell Genomics

Article Title: Recurrent breakpoints in the BRD4 locus reduce toxicity associated with gene amplification

doi: 10.1016/j.xgen.2025.100815

Figure Lengend Snippet: Use of CRISPR-Cas9 technology to mimic the effect of BRD4 focal deletions (A) Confirmation of Cas9 expression in OVSAHO cells by immunoblotting. (B) Fraction of GFP + cells detected in Cas9 activity assay. To measure Cas9 cutting efficiency, OVSAHO-Cas9 cells were transduced with a vector encoding GFP and sgGFP. After 10 days, a decreased population of GFP + cells was detected by flow cytometry, confirming Cas9 activity. (C) Intronic BRD4 regions targeted by CRISPR-Cas9 sgRNAs (inset not to scale due to length of intron 1). (D) CRISPR sequencing results for sgBRD4_region1 and sgBRD4_region2, showing the percentage of uncut, frameshift, and in-frame BRD4 reads when aligned to a reference sequence. (E) Expression of BRD4 isoforms following CRISPR-Cas9-mediated sgRNA cutting, relative to sgGFP control. Median and standard deviations of three replicates, two-way ANOVA. (F) Quantification of cell confluency in OVSAHO-Cas9 cells following CRISPR-Cas9-mediated sgRNA cutting of BRD4 regulatory regions or sgGFP control. Mean and standard deviation of three replicates, two-way ANOVA followed by Dunnett post-tests, controlling the Family-wise alpha threshold and confidence level. p values of the final time point are annotated. (G) Landscape of BRD4 dependency across ovarian cancers. Chronos scores (with mean and standard deviation) are shown for every ovarian cancer cell line included in the DepMap Public 23Q4 release. A score of −1 (median score for essential genes; red dashed line) is used as reference to indicate genetic dependency.

Article Snippet: After blocking with 5% dry milk in TBS-T for 30 min, the membrane was incubated at 4°C overnight with primary antibodies against rabbit BRD4 (cat. no. 702448, ThermoFisher Scientific), rabbit BRD4-L (cat. no. A301-985A100, Bethyl Laboratories), mouse SpCas9 (cat. no. 14697, Cell Signaling Technology), or rabbit GAPDH (cat. No. 2118, Cell Signaling Technology).

Techniques: CRISPR, Expressing, Western Blot, Activity Assay, Transduction, Plasmid Preparation, Flow Cytometry, Sequencing, Control, Standard Deviation

Journal: Cell Genomics

Article Title: Recurrent breakpoints in the BRD4 locus reduce toxicity associated with gene amplification

doi: 10.1016/j.xgen.2025.100815

Figure Lengend Snippet: Fine-tuned levels of BRD4 global expression are required for sustained cellular proliferation (A) Experimental workflow for mimicking concomitant focal BRD4 deletions occurring in BRD4 overexpressing ovarian cancer cells. (B) Rescue experiment by modulation of BRD4 expression. Quantification of cell confluency after CRISPR-Cas9-mediated gene ablation was measured in OVSAHO-Cas9 cells over-expressing BRD4-L (top) or BRD4-S(a) isoforms (bottom). Mean and standard error from three replicates, two-way ANOVA followed by Dunnett post-tests, controlling the Family-wise alpha threshold and confidence level. p values of the final time point are annotated. (C) Model of BRD4 expression regulation. In ovarian cancers, too-low or too-high expression levels are detrimental to cellular fitness. In BRD4 -amplified tumors, focal deletions serve as a mechanism to dampen global gene expression and rescue cellular proliferation.

Article Snippet: After blocking with 5% dry milk in TBS-T for 30 min, the membrane was incubated at 4°C overnight with primary antibodies against rabbit BRD4 (cat. no. 702448, ThermoFisher Scientific), rabbit BRD4-L (cat. no. A301-985A100, Bethyl Laboratories), mouse SpCas9 (cat. no. 14697, Cell Signaling Technology), or rabbit GAPDH (cat. No. 2118, Cell Signaling Technology).

Techniques: Expressing, CRISPR, Amplification, Gene Expression

Journal: Cell Genomics

Article Title: Recurrent breakpoints in the BRD4 locus reduce toxicity associated with gene amplification

doi: 10.1016/j.xgen.2025.100815

Figure Lengend Snippet:

Article Snippet: After blocking with 5% dry milk in TBS-T for 30 min, the membrane was incubated at 4°C overnight with primary antibodies against rabbit BRD4 (cat. no. 702448, ThermoFisher Scientific), rabbit BRD4-L (cat. no. A301-985A100, Bethyl Laboratories), mouse SpCas9 (cat. no. 14697, Cell Signaling Technology), or rabbit GAPDH (cat. No. 2118, Cell Signaling Technology).

Techniques: Virus, Expressing, Recombinant, Plasmid Preparation, Transfection, cDNA Synthesis, Bicinchoninic Acid Protein Assay, Software, CRISPR, Amplification, Sequencing