Journal: bioRxiv

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

doi: 10.64898/2026.01.31.702994

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

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

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

Journal: bioRxiv

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

doi: 10.64898/2026.01.31.702994

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

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

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

Journal: bioRxiv

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

doi: 10.64898/2026.01.31.702994

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

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

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

Journal: bioRxiv

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

doi: 10.64898/2026.01.31.702994

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

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

Techniques: Sequencing, Control

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gkaf1513

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

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

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

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gkaf1513

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

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

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

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gkaf1513

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

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

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

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gkaf1513

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

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

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

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gkaf1513

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

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

Techniques: Methylation, Binding Assay

Journal: bioRxiv

Article Title: PROTAC-Driven Protective Therapy increases the therapeutic window of anticancer drugs

doi: 10.64898/2026.01.12.698947

Figure Lengend Snippet: A) Structure of the MDM2-recruiting BRD4 degrader A1874 formed by JQ1 (a BRD4 inhibitor) and Idasanutlin (an MDM2 antagonist). B) BRD4, MDM2, p53 and p21 protein levels in isogenic TP53 WT and KO HCT116 cells treated with 1 µM A1874 for 24 h, assessed by western blot. Vinculin (VLC) was used as loading control. C) MDM2 mRNA expression levels in TP53 WT and TP53 KO HCT116 cells treated with 1 µM A1874 for 24 h assessed by qPCR. Error bars indicate mean ± s.d. (n = 3). D) Representative immunofluorescence images of MDM2 levels in TP53 WT and KO HCT116 cells upon treatment with vehicle or 1 µM A1874 for 24 h. Quantification of MDM2 nuclear intensity from 1600 cells is shown on the right. Statistical significance was determined with unpaired t-tests. **** = p < 0.0001.

Article Snippet: Cells were treated with the following compounds at the indicated doses: BRD4 PROTAC A1874 (HY-114305, MedChemExpress) at 1 μM; PARP1 PROTACs SK-575 (HY-139156, MedChemExpress) at 100 nM and 180055 (HY-170620, MedChemExpress) at 1 μM; and PARP inhibitors Talazoparib (HY-16106, MedChemExpress) and Olaparib (HY-10162, MedChemExpress) at concentrations ranging from 48 μM to 0.02 μM in 3-fold dilutions.

Techniques: Western Blot, Control, Expressing, Immunofluorescence

Journal: bioRxiv

Article Title: PROTAC-Driven Protective Therapy increases the therapeutic window of anticancer drugs

doi: 10.64898/2026.01.12.698947

Figure Lengend Snippet: A) BRD4, MDM2, p53, and p21 protein levels in a panel of cancer cell lines treated with vehicle or 1 µM A1874 for 24 h, assessed by western blot. Vinculin was used as loading control. Left panel displays colon cancer lines, including TP53 WT (HCT116 and RKO) and mutant (DLD1 and HT29) cells. Middle panel displays ovarian cancer cell lines, including TP53 WT (A2780) and mutant (ES2, OVCAR8 and SKOV3) cells. Right panel displays BJ and RPE1 p53-proficient primary cell lines. B) MDM2 mRNA expression levels in the indicated cell lines treated with vehicle or 1 µM A1874 for 24 h, assessed by qPCR. Error bars indicate mean ± s.d. (n = 3). C) Schematic representation of the CRISPRa/dCas9-SAM system. It consists of a dead Cas9 (dCas9) fused to the transcriptional activator VP64. The gRNA targeting MDM2 promoter forms a complex with MS2 (blue) and recruits transcriptional p65 (orange) and HSF1 (red) activators. This induces MDM2 expression from the endogenous locus. D) MDM2 mRNA expression levels in SKOV3 transduced with E.V. or gRNAs targeting MDM2 promoter, assessed by qPCR. Error bars indicate mean ± s.d. (n = 3). E) BRD4, and MDM2 protein levels in empty vector (E.V.)-transduced or MDM2-overexpressing SKOV3 cell lines treated with 1 µM A1874 for 24 h, assessed by western blot. Vinculin was used as loading control. F) MDM2 mRNA expression levels in the indicated cells treated with vehicle or 1 µM A1874 for 24 h, assessed by qPCR. Error bars indicate mean ± s.d. (n = 3).

Article Snippet: Cells were treated with the following compounds at the indicated doses: BRD4 PROTAC A1874 (HY-114305, MedChemExpress) at 1 μM; PARP1 PROTACs SK-575 (HY-139156, MedChemExpress) at 100 nM and 180055 (HY-170620, MedChemExpress) at 1 μM; and PARP inhibitors Talazoparib (HY-16106, MedChemExpress) and Olaparib (HY-10162, MedChemExpress) at concentrations ranging from 48 μM to 0.02 μM in 3-fold dilutions.

Techniques: Western Blot, Control, Mutagenesis, Expressing, Transduction, Plasmid Preparation

Journal: Cell Reports Medicine

Article Title: A triple-action PROTAC for wild-type p53 cancer therapy

doi: 10.1016/j.xcrm.2025.102467

Figure Lengend Snippet: Design and in vitro activity of TAPTAC1 (A) A chimera composed of a stapled p53 peptide and a small molecule BET protein inhibitor was designed to achieve triple-action targeting of HDM2, HDMX, and BET proteins to maximally restore p53 while hijacking HDM2 to degrade an oncogenic driver (BET proteins) instead of a tumor suppressor (p53). (B) Chemical composition of TAPTAC1, which links the BET inhibitor JQ1 to the stapled p53 peptide via a lysine-βAla moiety installed at position 25 of the p53 transactivation helix. (C) MD simulations demonstrate the assembly of a ternary complex between TAPTAC1 and the respective JQ1- and p53-binding domains of BRD4 and HDM2. (D) TAPTAC1 effectively generated ternary complexes (green) between the BD1 domain of BRD4 and HDM2 (left) and HDMX (right). Control elution profiles are shown for the individual proteins alone, including BRD4 BD1 (cyan), HDM2 (red), and HDMX (orange), and their combinations, including BRD4 BD1 and HDM2 (purple) and BRD4 BD1 and HDMX (brown). Each SEC experiment was repeated twice using independent preparations of proteins with similar results. (E) An in vitro ubiquitylation assay demonstrated the natural selectivity of HDM2 for p53, as evidenced by time-dependent laddering of p53 but not BRD4 BD1-BD2 (left 4 lanes). In the presence of TAPTAC1, the primary target of HDM2 is switched from p53 to BRD4 BD1-BD2 , which exhibits newfound laddering at the expense of p53 (right 4 lanes). Ubiquitylation assays were repeated three times with independent preparations of proteins and reagents with similar results. See also and .

Article Snippet: Recombinant FLAG-p53 (20 μM, BPS Bioscience, 100412) and recombinant 6xHis-BRD4 bearing BD1 and BD2 domains (20 μM, BPS Bioscience, 31045) were added as substrates for the ubiquitylation assay, in the absence or presence of TAPTAC1 (60 μM), and incubated for 0, 1, 2, or 4 h. Reactions were stopped with LDS buffer (Invitrogen) and boiled at 95°C for 5 min.

Techniques: In Vitro, Activity Assay, Binding Assay, Generated, Control, Ubiquitin Assay

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