Journal: Drug Design, Development and Therapy

Article Title: The Effect of AZD5153 on Radiosensitivity in Pancreatic Cancer Cells Through ATM-chk1 Pathway

doi: 10.2147/DDDT.S568551

Figure Lengend Snippet: Effect of AZD5153 on protein expression in irradiated pancreatic cancer cells, n=6. ( a and b ) Western blot analysis was performed to detect the expression of BRD4, c-Myc in different groups, β-actin was used as a loading control. ( c and d ) The protein expression of p-ATM, ATM, p-chk1, chk1, p-cdc25, cdc25, p-cdc2, cdc2 in different groups was detected by western blot, β-actin was used as a loading control. Results shown are the means ± SD of 3 independent experiments with similar results for all assays. Significance was determined by Student’s t -test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The membranes were blocked with 5% skim milk for 2 h, and incubated with primary antibodies against BRD4 (Cell Signaling Technology, Cat# 13440s), c-Myc (Cell Signaling Technology, Cat# 18583s), p-ATM (Abcam, Cat# ab81292), ATM (Cell Signaling Technology, Cat# 2873s), p-cdc25C (Cell Signaling Technology, Cat# 4901s), cdc25C (Cell Signaling Technology, Cat# 4688s), p-chk1 (Cell Signaling Technology, Cat# 2348s), chk1 (Abcam, Cat# ab40866), p-cdc2 (Cell Signaling Technology, Cat# 4539s), cdc2 (Cell Signaling Technology, Cat# 9116s), γ-H2AX (Cell Signaling Technology, Cat# 9718s), cleaved PARP (Cell Signaling Technology, Cat# 5625s), Bax (Cell Signaling Technology, Cat# 14796s), β-actin (Abcam, Cat# ab6276),Vinculin (Cell Signaling Technology, Cat# 13901s) overnight at 4 °C.

Techniques: Expressing, Irradiation, Western Blot, Control

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: Signal Transduction and Targeted Therapy

Article Title: Employing epigenetic protein degradation techniques to block CCL5-mediated photodynamic therapy via a programmed delivery platform

doi: 10.1038/s41392-025-02542-y

Figure Lengend Snippet: In vitro antitumor effects of ARV/Ce6@RDP on cell proliferation, apoptosis, and the cell cycle. a Schematic of the antitumor mechanism in cells via PDT and BRD4 PROTAC mediated by ARV/Ce6@RDP. The figure was created with Figdraw.com. b Representative images of ROS production in 4T1 and B16F10 cells subjected to different treatments (blue: nucleus; green: DCFH-DA-labeled ROS). Scale bar: 100 µm. c Flow cytometric analyses of ROS production in 4T1 and B16F10 cells after various treatments ( n = 3 per group, two-tailed unpaired Student’s t -test). d Western blot analysis of BRD4 and c-Myc protein expression in 4T1 and B16F10 cells after various treatments. e Viability of 4T1 and B16F10 cells following different treatments ( n = 3 per group, two-tailed unpaired Student’s t -test). f Flow cytometric analysis of apoptosis in 4T1 and B16F10 cells after different treatments by Annexin V-FITC/PI double staining ( n = 3 per group, two-tailed unpaired Student’s t -test). g Flow cytometric analysis of the cell cycle in 4T1 and B16F10 cells receiving different treatments by PI staining ( n = 3 per group). h Changes in p-AKT, AKT, p-ERK1/2, ERK1/2, p-STAT3, and STAT3 protein levels in 4T1 and B16F10 cells following various treatments. i Changes in the protein levels of genes involved in the apoptosis pathway after various treatments in 4T1 and B16F10 cells. j Changes in the protein levels associated with the cell cycle in 4T1 and B16F10 cells following various treatments. All the data in this figure are presented as the means ± SDs

Article Snippet: Chromatin samples were precipitated with rabbit anti-BRD4 antibody (#83375, CST) or an equivalent amount of normal rabbit IgG isotype control (#2729, CST) prior to the addition of protein G magnetic beads.

Techniques: In Vitro, Labeling, Two Tailed Test, Western Blot, Expressing, Double Staining, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: Employing epigenetic protein degradation techniques to block CCL5-mediated photodynamic therapy via a programmed delivery platform

doi: 10.1038/s41392-025-02542-y

Figure Lengend Snippet: Mechanistic study of the antitumor effects of ARV/Ce6@RDP micelles. a Venn diagrams of the intersection between upregulated genes in Ce6@RDP (+) vs RDP and downregulated genes in ARV/Ce6@RDP (+) vs Ce6@RDP (+) for 4T1 and B16F10 cells based on RNA-seq analysis. b Venn diagrams of the intersection between upregulated genes in Ce6@RDP (+) vs RDP and upregulated genes in ARV/Ce6@RDP (+) vs Ce6@RDP (+) for 4T1 and B16F10 cells. c Heatmap of the intersection genes from ( a ) and ( b ) expressed in 4T1 and B16F10 cells receiving different treatments. d Quantification of the CCL5 level in the supernatants of treated 4T1 and B16F10 cells ( n = 3 per group, two-tailed unpaired Student’s t -test). Cell viability ( e ) and apoptosis ( f ) analysis of Ccl5 -knockdown 4T1 and B16F10 cells with and without treatment with PDT ( n = 3 per group, two-tailed unpaired Student’s t -test). g – n Effects of Ccl5 knockdown on PDT efficacy in vivo. Tumor growth curves ( g , n = 6 per group, two-way ANOVA with Tukey’s multiple comparisons test), post-treatment tumor photographs ( h ), and tumor weight analysis ( i , n = 6 per group, two-tailed unpaired Student’s t -test) of Ccl5 -knockdown 4T1 cells and control cell xenografts treated with or without PDT; Tumor growth curves ( j , n = 5 per group, two-way ANOVA with Tukey’s multiple comparisons test), post-treatment tumor photographs ( k ) and tumor weight analysis ( l , n = 5 per group, two-tailed unpaired Student’s t -test) of Ccl5 -knockdown B16F10 cells and control cell xenografts treated with or without PDT; Expression levels of the CCL5 protein ( m ) and proteins ( n ) related to proliferation and apoptosis in tumor tissues collected from Ccl5 -knockdown 4T1 and B16F10 xenografts after treatment with or without PDT. o Flow cytometric analyses of M2 polarization in BMDMs after treatment with culture medium from Ccl5 -knockdown 4T1 cells or control cells ( n = 3 per group, two-tailed unpaired Student’s t -test). p M2 polarization analysis of BMDMs treated with culture medium from Ccl5 -overexpressing 4T1 cells and control cells ( n = 3 per group, two-tailed unpaired Student’s t -test). q Mrc1 , Arg1 , Irf4 , Ym1 , and Cd274 gene expression in BMDMs incubated with culture medium from different 4T1 cells determined by qPCR ( n = 3 per group, two-tailed unpaired Student’s t -test). r Recruitment of M2 macrophages by Ccl5 -knockdown or Ccl5 -overexpressing 4T1 cells ( n = 3 per group; two-tailed unpaired Student’s t -test; scale bar: 50 µm). s CFSE staining analysis of 4T1 cell proliferation in BMDMs subjected to different treatments ( n = 3 per group, two-tailed unpaired Student’s t -test). t Effects of nanomedicines on Ccl5 promoter activity detected by dual-luciferase assay ( n = 3 per group, two-tailed unpaired Student’s t -test). u ChIP‒qPCR analysis of BRD4 protein binding to the Ccl5 promoter in B16F10 cells ( n = 3 per group; two-way ANOVA with Sidak’s multiple comparisons test). v Schematic diagram of ARV-825-mediated inhibition of Ccl5 gene transcription to enhance PDT. The figure was created with Figdraw.com. The data are presented as the means ± SDs for in vitro experiments and means ± SEMs for in vivo experiments

Article Snippet: Chromatin samples were precipitated with rabbit anti-BRD4 antibody (#83375, CST) or an equivalent amount of normal rabbit IgG isotype control (#2729, CST) prior to the addition of protein G magnetic beads.

Techniques: RNA Sequencing, Two Tailed Test, Knockdown, In Vivo, Control, Expressing, Gene Expression, Incubation, Staining, Activity Assay, Luciferase, Protein Binding, Inhibition, In Vitro

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

Journal: bioRxiv

Article Title: Tuning the open-close equilibrium of Cereblon with small molecules influences protein degradation

doi: 10.64898/2025.12.19.695617

Figure Lengend Snippet: A) Structural analysis of closed CRBN and lenalidomide, showing the network of hydrogen bonds in 5FQD. The structure is coloured by domain Lon (yellow) (N-terminal belt (purple), HB (orange), TBD (salmon), (sensor loop (teal)). Lenalidomide is shown as black sticks, the Tryptophan cage and residues involved in hydrogen bonds are shown in stick form, hydrogen bonds are represented by dashed grey lines. B) A schematic representation of the network of interactions. Backbone to sidechain H-bonds are represented by solid lines, sidechain to sidechain H-bonds by dotted lines and ligand to protein H-bonds by arrows. C) Turbidity first derivative of thermal denaturation for CRBN midi WT and mutants in the absence (black) or presence of binders: lenalidomide (red), 4 (grey), 8 (cyan) and 9 (blue). WT is mean of triplicates. D) Dimensionless Kratky plot and table of Rg values generated from SAXS data of CRBN midi and CRBN midi mutants in complex with dihydrouracil 9 . E) Representative Western Blot assessing BRD4 degradation efficiency in DLD-1 WT cells, DLD-1 CRBN KO cells and DLD-1 CRBN KO cells stably expressing FLAG-CRBN WT or CRBN point mutants. Cells were treated with dBET6 (1 µM) for 18h and degradation of BRD4 long and short isoform was quantified and normalised against a loading control and DMSO-treated samples. F) Quantification of the bands from (E).

Article Snippet: Membranes were probed with primary antibodies targeting BRD4 ( # 1:1000), CRBN (CST; #71810, 1:1000).

Techniques: Generated, Western Blot, Stable Transfection, Expressing, Control

Journal: Scientific Reports

Article Title: Overexpression of JMJD6 drives immune evasion via the BRD4–IRF1–PD-L1 axis and promotes malignancy in gastric cancer

doi: 10.1038/s41598-025-30705-y

Figure Lengend Snippet: Graphical abstract. Graphical abstract illustrating the hypothetical mechanism by which JMJD6 promotes tumor progression and immune evasion in GC. JMJD6 is overexpressed in gastric cancer cells and promotes BRD4 expression, which upregulates IRF1 and consequently increases PD-L1 expression. Elevated PD-L1 expression on tumor cells inhibits T cell–mediated antitumor immunity, thereby facilitating immune escape.

Article Snippet: Anti-JMJD6 mouse monoclonal antibody (sc-28348; Santa Cruz Biotechnology, TX, USA), anti-PD-L1 rabbit monoclonal antibody (13684; Cell Signaling Technology, MA, USA), anti-ACTB rabbit monoclonal antibody (3700; Cell Signaling Technology), anti-BRD4 rabbit polyclonal antibody (A301-985A50; Bethyl Laboratories, TX, USA), and anti-IRF1 rabbit monoclonal antibody (8478; Cell Signaling Technology) were used.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Overexpression of JMJD6 drives immune evasion via the BRD4–IRF1–PD-L1 axis and promotes malignancy in gastric cancer

doi: 10.1038/s41598-025-30705-y

Figure Lengend Snippet: JMJD6 regulates BRD4, IRF1 and PD-L1 expression. ( a ) The knockdown of JMJD6 by transfection with siRNA-JMJD6 suppressed BRD4, IRF1 and PD-L1 in MKN74. In addition, the knockdown of BRD4 by transfection with siRNA-BRD4 suppressed IRF1 and PD-L1 in MKN74. In contrast, the knockdown of BRD4 did not suppress JMJD6 in MKN74. ( b ) Knockdown of JMJD6 suppressed PD-L1 and BRD4 expression in MKN74 gastric cancer (GC) cells. White dotted lines indicate nuclear boundaries. ( c ) Co-culture assay of GC cells and T cells. Under JMJD6 knockdown, T cells had more potent anti-tumor activity against GC cells compared with NC, and the proliferation ratio of GC cells was significantly decreased (mean ± SD, n = 3; error bars indicate SD, n = 3). ( d ) An impedance-based tumor-cell killing assay. The knockdown of JMJD6 increased the anti-tumor activity of T cells and inhibited the proliferation of GC cells. ( e ) JMJD6 overexpression using plasmid transfection promotes BRD4, IRF1 and PD-L1 expression. ( f ) A hypothetical model of the overexpression or activation of JMJD6 in GC cells.

Article Snippet: Anti-JMJD6 mouse monoclonal antibody (sc-28348; Santa Cruz Biotechnology, TX, USA), anti-PD-L1 rabbit monoclonal antibody (13684; Cell Signaling Technology, MA, USA), anti-ACTB rabbit monoclonal antibody (3700; Cell Signaling Technology), anti-BRD4 rabbit polyclonal antibody (A301-985A50; Bethyl Laboratories, TX, USA), and anti-IRF1 rabbit monoclonal antibody (8478; Cell Signaling Technology) were used.

Techniques: Expressing, Knockdown, Transfection, Co-culture Assay, Activity Assay, Over Expression, Plasmid Preparation, Activation Assay