Journal: bioRxiv

Article Title: R egulated Induced Proximity Targeting Chimeras (RIPTACs): a Novel Heterobifunctional Small Molecule Therapeutic Strategy for Killing Cancer Cells Selectively

doi: 10.1101/2023.01.01.522436

Figure Lengend Snippet: a) 3h treatment of 293_HFL cells with indicated compounds followed by HaloTag immunoprecipitation using HaloTrap beads demonstrates cellular ternary complex formation with both the non-covalent RIPTAC HLDA-222 and the covalent RIPTAC HLDA-121, but not with the control molecule HLDA-125 that does not bind BRD4. b) Ternary complex formation with HLDA-121 and HLDA-222 can be competed away with 30min pre-treatment with TAMRA-CA and HLDA-001, respectively. 30min JQ1 pre-treatment competes away complex formation with both RIPTACs partially. c) 293_HFL cells were treated with the indicated compounds for 3h (T 0 ), following which the compound treated medium was washout out and replaced with normal growth medium for up to 72h. The HaloTag-FKBP fusion protein was immunoprecipitated at the timepoints shown and the BRD4 protein levels in the complex were detected by immunoblotting.d) AlphaLISA assay measuring BRD4-BD1 inhibition demonstrates positive co-operativity in biochemical RIPTAC ternary complex formation in presence of the non-covalent RIPTAC HLDA-221 and FKBP. e) 7 day CellTiter Glo viability assay with indicated covalent RIPTACs in cell lines expressing the HaloTag-FKBP fusion protein selectively in the nucleus (293_NLS2HF), plasma membrane (293_MYRHF), or both (293_HFL). f) 7 day CellTiter Glo viability assay in the same cell lines using non-covalent RIPTACs.

Article Snippet: Immunoblotting was done using mouse-anti FKBP (#sc-136962, Santa cruz), rabbit-anti BRD4 (#13440S, Cell Signaling), mouse-anti Plk1 (#17056, Abcam) and rabbit-anti CDK9 (#2316, Cell signaling), rabbit-anti phospho-Rpb1 CTD Ser2 (#13499, Cell Signaling)

Techniques: Immunoprecipitation, Control, Western Blot, Inhibition, Viability Assay, Expressing, Clinical Proteomics, Membrane

Journal: Redox Biology

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

doi: 10.1016/j.redox.2016.12.031

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

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

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

Journal: Redox Biology

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

doi: 10.1016/j.redox.2016.12.031

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

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

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

Journal: Redox Biology

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

doi: 10.1016/j.redox.2016.12.031

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

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

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

Journal: Redox Biology

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

doi: 10.1016/j.redox.2016.12.031

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

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

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

Journal: Frontiers in Medicine

Article Title: Correlation of Bromodomain Protein BRD4 Expression With Epithelial–Mesenchymal Transition and Disease Severity in Chronic Rhinosinusitis With Nasal Polyps

doi: 10.3389/fmed.2020.00413

Figure Lengend Snippet: The sequences of PCR primers used in this study.

Article Snippet: Rabbit polyclonal anti-BRD4 antibody (ab128874, 1:100 diluted) was from Abcam.

Techniques:

Journal: Frontiers in Medicine

Article Title: Correlation of Bromodomain Protein BRD4 Expression With Epithelial–Mesenchymal Transition and Disease Severity in Chronic Rhinosinusitis With Nasal Polyps

doi: 10.3389/fmed.2020.00413

Figure Lengend Snippet: (A) Immunofluorescence staining (DAPI) on 20-μm slides from fresh frozen NP tissue (CRSwNPs) and control inferior turbinate mucosa (healthy control) performed with polyclonal antibodies against rabbit BRD4. (B) The relative mRNA expression of BRD4 in tissue homogenates from patients with CRSwNP ( n = 24) and controls ( n = 24) after qRT-PCR analysis normalized to the housekeeping gene GAPDH. (C) Plot of EMT marker E-cadherin, vimentin, and Snai1 in CRSwNPs and healthy control. (D) The relative mRNA expression of BRD4 after qRT-PCR analysis in primary hNPDECs and hNECs isolated and cultured from NP tissues ( n = 7) and healthy nasal inferior turbinate tissues ( n = 7). Data were expressed as mean ± SD. The Student test was used for statistical comparison. * P < 0.05, ** P < 0.01 vs. controls.

Article Snippet: Rabbit polyclonal anti-BRD4 antibody (ab128874, 1:100 diluted) was from Abcam.

Techniques: Immunofluorescence, Staining, Control, Expressing, Quantitative RT-PCR, Marker, Isolation, Cell Culture, Comparison

Journal: Frontiers in Medicine

Article Title: Correlation of Bromodomain Protein BRD4 Expression With Epithelial–Mesenchymal Transition and Disease Severity in Chronic Rhinosinusitis With Nasal Polyps

doi: 10.3389/fmed.2020.00413

Figure Lengend Snippet: Effect of BRD4 silencing on TGF-β1–induced EMT marker production (E-cadherin, vimentin, and Snai1) production in human primary hNPDECs (A–C) . Human primary hNPDECs were either transfected with siBRD4 or siCONT. Effect of BRD4 inhibitor JQ1 on TGF-β1 induction EMT marker production in human primary hNPDECs (D–F) . E-cadherin, vimentin, and Snai1 mRNA expression was assessed by qRT-PCR. Fold change was calculated relative to siCONT–TGF-β1. All data are displayed as mean ± SD. The one-way ANOVA was used for statistical comparison. * P < 0.05, ** P < 0.01 vs. controls.

Article Snippet: Rabbit polyclonal anti-BRD4 antibody (ab128874, 1:100 diluted) was from Abcam.

Techniques: Marker, Transfection, Expressing, Quantitative RT-PCR, Comparison

Journal: Frontiers in Medicine

Article Title: Correlation of Bromodomain Protein BRD4 Expression With Epithelial–Mesenchymal Transition and Disease Severity in Chronic Rhinosinusitis With Nasal Polyps

doi: 10.3389/fmed.2020.00413

Figure Lengend Snippet: (A) Correlation between CT scan score (Lund–Mackay) and BRD4 mRNA expression in CRSwNP ( n = 24). (B–D) The association between CT scan score and E-cadherin, vimentin, and Snai1 mRNA expression in CRSwNP ( n = 24) was presented. The Spearman rank correlation coefficient was used for statistical comparison.

Article Snippet: Rabbit polyclonal anti-BRD4 antibody (ab128874, 1:100 diluted) was from Abcam.

Techniques: Computed Tomography, Expressing, Comparison

Journal: iScience

Article Title: Profiling of diverse tumor types establishes the broad utility of VHL-based ProTaCs and triages candidate ubiquitin ligases

doi: 10.1016/j.isci.2022.103985

Figure Lengend Snippet:

Article Snippet: MSD Detection Ab, Mouse mAb , Atlas Antibodies , Cat# AMAb90841 lots 03087, 02741, & 02739; RRID: AB_2665685.

Techniques: Nucleic Acid Electrophoresis, Electrophoresis, Software, Simple Western

Journal: bioRxiv

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

doi: 10.64898/2026.01.31.702994

Figure Lengend Snippet: a and b ) Immunoblots showing depletion of BRD4-L and BRD4-S after 4 hours of ZxH-3-26 treatment (ZxH, BRD4-specific PROTAC) (a); dTAGV-1 and dTAG13 mediated BRD4 degradation in BRD4-dTAG hESCs (b), BRD3 and β-ACTIN serve as controls. c ) Time-course heatmap of RNA-seq data (4 hours, 8 hours, 20 hours) of PROTAC treatment and 20 hours of dTAGV-1 treatment in BRD4-dTAG hESCs comparing log2fold change values across four k-means clusters (C1–C4) based on differential expression levels, indicating similar directional changes at least in two of the ZxH treatment time points (left). Heatmaps of CUT&Tag counts per million reads (CPM) signal for short and long isoforms of BRD4 (Diagenode and Abcam antibodies) (middle). Enrichment of GO biological processes of the genes in the four clusters (right). d ) Genome-browser visualization of CUT&Tag for BRD4 performed using two antibodies, along with average RNAseq signal (n=3 replicates), performed 8 hours after DMSO and ZxH treatment in H9 hESCs at representative neuronal and developmental genes, along with known BRD4 target gene MYC. e ) Percentage peak overlap for BRD4, EED, RAD21, NIPBL, serine-5 phosphorylated RNA Pol II (RNA-Pol II s5p), H3K27ac, H3K4me3, and H3K27me3 across 15 ChromHMM states in H9-hESCs.

Article Snippet: The precleared extract was then rotated with 1 μg of antibodies anti-BRD4 (Cell Signalling, Cat. # 83375), anti-RING1B (Active Motif, Cat. # 39663), anti-MGA (Antibodies Online, Cat.# ABIN2444597) or IGG (ThermoFisher Scientific, Cat. # 500-P00-500UG) for 2 hours at 4 °C before adding the Dynabeads A bead for 30min at 4 °C.

Techniques: Western Blot, RNA Sequencing, Quantitative Proteomics

Journal: bioRxiv

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

doi: 10.64898/2026.01.31.702994

Figure Lengend Snippet: a) Pairwise peak intersection for chromatin modifications. Values indicate the fraction of overlap between peak-sets. Horizontal comparison shows the percentage of overlap between each peak set on the X-axis, with peak sets compared on the Y-axis, and vice versa. b ) Heatmaps of CUT&Tag counts per million reads (CPM) signal for BRD4 (BRD4, Diagenode antibody), BRD4(Abcam antibody), H3K27me3, H3K4me3, H3K27ac, CUT&RUN for BRD2, BRD3, EED and EZH2, ChIPseq data for PRC1.6 components (PCGF6, MAX, MYC), along with PRC1 component (CBX8 and RING1B). Clustered based on enrichment of PRC1.6 components, active (H3K4me3), bivalent (H3K27me3+ & H3K4me3+), and other gene promoters. c ) Venn diagrams and Metascape functional annotations (below) of upregulated (left, in purple) and downregulated (right, in purple) genes following 8 hours of ZxH-mediated BRD4 degradation and in two PCGF6 knockout human pluripotent stem cell lines (data from Lan et.al. 2022). d ) Similar to (b), but clustering based on commonly upregulated genes (clusters 1-3). Upregulated gene promoters are categorized by their bivalent or active chromatin modifications. e ) Genome-browser visualization of BRD4, MAX, and bivalent histone modifications, along with average TTseq signal (n=3 replicates), performed 1 hour after DMSO and dTAGV-1 treatment in BRD4-dTAG hESCs (Western blotting showing BRD4 degradation in ).

Article Snippet: The precleared extract was then rotated with 1 μg of antibodies anti-BRD4 (Cell Signalling, Cat. # 83375), anti-RING1B (Active Motif, Cat. # 39663), anti-MGA (Antibodies Online, Cat.# ABIN2444597) or IGG (ThermoFisher Scientific, Cat. # 500-P00-500UG) for 2 hours at 4 °C before adding the Dynabeads A bead for 30min at 4 °C.

Techniques: Comparison, Functional Assay, Knock-Out, Western Blot

Journal: bioRxiv

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

doi: 10.64898/2026.01.31.702994

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

Article Snippet: The precleared extract was then rotated with 1 μg of antibodies anti-BRD4 (Cell Signalling, Cat. # 83375), anti-RING1B (Active Motif, Cat. # 39663), anti-MGA (Antibodies Online, Cat.# ABIN2444597) or IGG (ThermoFisher Scientific, Cat. # 500-P00-500UG) for 2 hours at 4 °C before adding the Dynabeads A bead for 30min at 4 °C.

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

Journal: bioRxiv

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

doi: 10.64898/2026.01.31.702994

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

Article Snippet: The precleared extract was then rotated with 1 μg of antibodies anti-BRD4 (Cell Signalling, Cat. # 83375), anti-RING1B (Active Motif, Cat. # 39663), anti-MGA (Antibodies Online, Cat.# ABIN2444597) or IGG (ThermoFisher Scientific, Cat. # 500-P00-500UG) for 2 hours at 4 °C before adding the Dynabeads A bead for 30min at 4 °C.

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

Journal: bioRxiv

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

doi: 10.64898/2026.01.31.702994

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

Article Snippet: The precleared extract was then rotated with 1 μg of antibodies anti-BRD4 (Cell Signalling, Cat. # 83375), anti-RING1B (Active Motif, Cat. # 39663), anti-MGA (Antibodies Online, Cat.# ABIN2444597) or IGG (ThermoFisher Scientific, Cat. # 500-P00-500UG) for 2 hours at 4 °C before adding the Dynabeads A bead for 30min at 4 °C.

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

Journal: bioRxiv

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

doi: 10.64898/2026.01.31.702994

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

Article Snippet: The precleared extract was then rotated with 1 μg of antibodies anti-BRD4 (Cell Signalling, Cat. # 83375), anti-RING1B (Active Motif, Cat. # 39663), anti-MGA (Antibodies Online, Cat.# ABIN2444597) or IGG (ThermoFisher Scientific, Cat. # 500-P00-500UG) for 2 hours at 4 °C before adding the Dynabeads A bead for 30min at 4 °C.

Techniques: Sequencing, Control

Journal: Current Issues in Molecular Biology

Article Title: Role of the Super-Enhancer Component Bromodomain Protein 4 in the Radiation Response of Human Head and Neck Squamous Cell Carcinoma Cells

doi: 10.3390/cimb48010071

Figure Lengend Snippet: Effect of ionizing radiation on the proliferation and apoptosis of ARV-771–treated head and neck squamous cell carcinoma (HNSCC) cells. ( A ) ARV-771–treated SAS and Ca9-22 cells for 24 h were collected and analyzed for bromodomain protein 4 (BRD4) protein expression using Western blotting. ( B – D ) ARV-771–treated cells were irradiated with 6 Gy and cultured for 3 days. Cells were counted, reseeded, and cultured for another 3 days (total, 6 days). The cultured cells for 3 or 6 days were harvested for estimation of proliferation ( B ) and apoptosis analysis ( C , D ). ( B ) Results show the cell proliferation rate calculated from the ratio of the number of viable cells at 3 (for SAS) or 6 days (for Ca9-22) of culture to the number of seeded cells. * p < 0.05. ( C ) Representative cytogram of Annexin V/propidium iodide staining in SAS cells are shown. ( D ) Results are shown as percentage of Annexin V-positive cells. * p < 0.05.

Article Snippet: Anti-BRD4 rabbit antibody (#63759), anti-deltaN p63 (E6Q3O) rabbit antibody (##67825), anti-Glyceraldehyde–3–phosphate dehydrogenase (GAPDH) rabbit antibody (#5174), anti-p-histone H2A.X (γH2AX; #9718), horseradish peroxidase-conjugated anti-rabbit IgG antibody (#7074), horseradish peroxidase-conjugated anti-mouse IgG antibody (#7076), and AlexaFluor 488 ® -conjugated mouse IgG antibody (#4408) were purchased from Cell Signaling Technology Japan, K.K. (Tokyo, Japan).

Techniques: Expressing, Western Blot, Irradiation, Cell Culture, Staining

Journal: Current Issues in Molecular Biology

Article Title: Role of the Super-Enhancer Component Bromodomain Protein 4 in the Radiation Response of Human Head and Neck Squamous Cell Carcinoma Cells

doi: 10.3390/cimb48010071

Figure Lengend Snippet: Effects of ARV-771 or BRD4 knockdown on radiosensitivity of HNSCC cells. ( A ) ARV-771–treated HNSCC cells were irradiated with X-rays, and cells were collected at 24 h following irradiation for colony assay. Results show survival rates at each dose with respect to non-irradiated cells. * p < 0.05. ( B ) SAS and Ca9-22 cells transfected with siRNA targeting BRD4 were harvested for BRD4 protein expression analysis by Western blotting. ( C ) BRD4-knockdown HNSCC cells were irradiated with X-rays, and cells were collected at 24 h following irradiation for colony assay. Results show survival rates at each dose with respect to non-irradiated cells. * p < 0.05.

Article Snippet: Anti-BRD4 rabbit antibody (#63759), anti-deltaN p63 (E6Q3O) rabbit antibody (##67825), anti-Glyceraldehyde–3–phosphate dehydrogenase (GAPDH) rabbit antibody (#5174), anti-p-histone H2A.X (γH2AX; #9718), horseradish peroxidase-conjugated anti-rabbit IgG antibody (#7074), horseradish peroxidase-conjugated anti-mouse IgG antibody (#7076), and AlexaFluor 488 ® -conjugated mouse IgG antibody (#4408) were purchased from Cell Signaling Technology Japan, K.K. (Tokyo, Japan).

Techniques: Knockdown, Irradiation, Colony Assay, Transfection, Expressing, Western Blot

Journal: Current Issues in Molecular Biology

Article Title: Role of the Super-Enhancer Component Bromodomain Protein 4 in the Radiation Response of Human Head and Neck Squamous Cell Carcinoma Cells

doi: 10.3390/cimb48010071

Figure Lengend Snippet: γH2AX and BRD4 localization and effects of ARV-771 or BRD4 knockdown γH2AX expression in irradiated SAS cells. ( A , B ) SAS cells were irradiated with 2 Gy and cultured for 30 min. Samples are analyzed using immunofluorescence staining. ( A ) DAPI (nuclei) and γH2AX in non-irradiated and 2 Gy–irradiated SAS cells. ( B ) Expression of BRD4 (green) and γH2AX (red) in 2 Gy–irradiated SAS cells. The white dotted line in the figure indicates the outline of the nucleus. ( C , D ) ARV-771–treated ( C ) or BRD4 knockdown ( D ) SAS cells were irradiated with X-rays, and cells were collected at 15 min, 30 min, and 3 h following irradiation for an analysis of γH2AX expression by Western blotting.

Article Snippet: Anti-BRD4 rabbit antibody (#63759), anti-deltaN p63 (E6Q3O) rabbit antibody (##67825), anti-Glyceraldehyde–3–phosphate dehydrogenase (GAPDH) rabbit antibody (#5174), anti-p-histone H2A.X (γH2AX; #9718), horseradish peroxidase-conjugated anti-rabbit IgG antibody (#7074), horseradish peroxidase-conjugated anti-mouse IgG antibody (#7076), and AlexaFluor 488 ® -conjugated mouse IgG antibody (#4408) were purchased from Cell Signaling Technology Japan, K.K. (Tokyo, Japan).

Techniques: Knockdown, Expressing, Irradiation, Cell Culture, Immunofluorescence, Staining, Western Blot

Journal: Current Issues in Molecular Biology

Article Title: Role of the Super-Enhancer Component Bromodomain Protein 4 in the Radiation Response of Human Head and Neck Squamous Cell Carcinoma Cells

doi: 10.3390/cimb48010071

Figure Lengend Snippet: γH2AX and BRD4 or ΔNp63 localization in irradiated SAS cells. SAS cells were irradiated with 2 Gy and cultured for 30 min. Samples were analyzed using immunofluorescence staining. ( Left panel ) Expression of BRD4 (green) and γH2AX (red) in 2 Gy–irradiated cells. ( Right panel ) Expression of ΔNp63 (green) and γH2AX (red) in 2 Gy–irradiated cells.

Article Snippet: Anti-BRD4 rabbit antibody (#63759), anti-deltaN p63 (E6Q3O) rabbit antibody (##67825), anti-Glyceraldehyde–3–phosphate dehydrogenase (GAPDH) rabbit antibody (#5174), anti-p-histone H2A.X (γH2AX; #9718), horseradish peroxidase-conjugated anti-rabbit IgG antibody (#7074), horseradish peroxidase-conjugated anti-mouse IgG antibody (#7076), and AlexaFluor 488 ® -conjugated mouse IgG antibody (#4408) were purchased from Cell Signaling Technology Japan, K.K. (Tokyo, Japan).

Techniques: Irradiation, Cell Culture, Immunofluorescence, Staining, Expressing

Journal: Current Issues in Molecular Biology

Article Title: Role of the Super-Enhancer Component Bromodomain Protein 4 in the Radiation Response of Human Head and Neck Squamous Cell Carcinoma Cells

doi: 10.3390/cimb48010071

Figure Lengend Snippet: Protein expression of BRD4 and ΔNp63α treated with ΔNp63 knockdown, ARV-771, and BRD4 knockdown cells. ( A ) ΔNp63 knockdown, ( B ) ARV-771–treated, ( C ) BRD4 knockdown SAS cells were harvested for Western blotting to analyze BRD4 and ΔNp63α protein expression.

Article Snippet: Anti-BRD4 rabbit antibody (#63759), anti-deltaN p63 (E6Q3O) rabbit antibody (##67825), anti-Glyceraldehyde–3–phosphate dehydrogenase (GAPDH) rabbit antibody (#5174), anti-p-histone H2A.X (γH2AX; #9718), horseradish peroxidase-conjugated anti-rabbit IgG antibody (#7074), horseradish peroxidase-conjugated anti-mouse IgG antibody (#7076), and AlexaFluor 488 ® -conjugated mouse IgG antibody (#4408) were purchased from Cell Signaling Technology Japan, K.K. (Tokyo, Japan).

Techniques: Expressing, Knockdown, Western Blot

Journal: Current Issues in Molecular Biology

Article Title: Role of the Super-Enhancer Component Bromodomain Protein 4 in the Radiation Response of Human Head and Neck Squamous Cell Carcinoma Cells

doi: 10.3390/cimb48010071

Figure Lengend Snippet: A proposed working model of BRD4-dependent regulation of DNA damage responses. Under a condition that BRD4 intact, BRD4-associated SEs support transcriptional programs involved in cell survival and DNA repair, contributing to efficient DNA damage repair and relative radioresistance. ARV-771 treatment induces pharmacological degradation of BRD4 and disorganization of SEs, leading to enhanced γH2AX signaling, transcriptional stress, and increased radiosensitization. In contrast, BRD4 knockdown alters SE function without inducing acute SE degradation, resulting in reduced γH2AX levels following irradiation and a distinct mode of radiosensitization. This model proposes that γH2AX reflects different biological processes depending on the mode of BRD4 suppression.

Article Snippet: Anti-BRD4 rabbit antibody (#63759), anti-deltaN p63 (E6Q3O) rabbit antibody (##67825), anti-Glyceraldehyde–3–phosphate dehydrogenase (GAPDH) rabbit antibody (#5174), anti-p-histone H2A.X (γH2AX; #9718), horseradish peroxidase-conjugated anti-rabbit IgG antibody (#7074), horseradish peroxidase-conjugated anti-mouse IgG antibody (#7076), and AlexaFluor 488 ® -conjugated mouse IgG antibody (#4408) were purchased from Cell Signaling Technology Japan, K.K. (Tokyo, Japan).

Techniques: Knockdown, Irradiation