brd4 protac arv 825  (MedChemExpress)

Journal: bioRxiv

Article Title: A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

doi: 10.1101/2025.09.06.674632

Figure Lengend Snippet: (A) Schematic showing the domain architecture of the Brd4 protein, and how its bromodomains are being used in several combinations to make acyl-eCRs and to determine how the valency of reader domains affects drug perturbations. (B) Immunofluorescence images of mESCs showing the nuclear localization of different valencies of the second bromodomain from BRD4 in the Parbit system (green) and their colocalization with Hoechst (magenta) after drug treatments. All scale bars are 5 µM. Drug treatments were performed at 1 μM concentrations for 24 hours. Bottom panel: Representative pseudocolored images (eGFP signal) depicting the differences in fluorescence intensities in different cell lines. A gradient pseudocolor bar (signal intensity) is shown at the left. (C) Normalized FACS data showing the effects of ARV-825 PROTAC treatment on cells expressing several combinations of bromodomains from BRD4. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

Article Snippet: The CBP/p300 bromodomain inhibitor: GNE-049 (MedChemExpress, HY-108435), CBP/p300 PROTAC: dCBP-1 (MedChemExpress, HY-134582), BRD4 bromodomain inhibitor: (+)-JQ-1 (MedChemExpress, HY-13030), BRD4 PROTAC: ARV-825 (MedChemExpress, HY-16954), BRD9 bromodomain inhibitor: iBRD9 (MedChemExpress, HY-18975), and broad-spectrum bromodomain inhibitor: Bromosporine (MedChemExpress, HY-15815) were dissolved in DMSO and then diluted to 1μM in mESC media for 24-hour treatments, unless stated otherwise.

Techniques: Immunofluorescence, Fluorescence, Expressing

Journal: bioRxiv

Article Title: A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

doi: 10.1101/2025.09.06.674632

Figure Lengend Snippet: (A) Top: Schematic showing how the competitive binding of small molecule inhibitors versus PROTACs for the binding pocket of Acyl-eCRs can be used to measure the affinity of a small molecule for a bromodomain in cellulo . Inhibitors with higher affinity for a bromodomain, better prevent PROTAC-induced degradation. Bottom : Treatment scheme for competitive binding experiments. Cells were treated with 1 μM inhibitors for 1 hour. Then, varying concentrations of the PROTAC were added in addition to the previously added inhibitor. After 3 hours of treatment, the cell fluorescence was measured via flow cytometry. (B) Competitive binding between ARV-825 and several small molecule inhibitors showing how the inhibitors bind to BRD4(2)_BRD.1x. The cells were treated with the indicated inhibitor at a 1 μM concentration for 1 hour. Then, the stated concentration of ARV-825 PROTAC was added for 3 hours, in addition to the previous concentration of the same inhibitor. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells. (C) Competitive binding between dCBP-1 and several small molecule inhibitors showing how the inhibitors bind CBP bromodomains in Acyl-eCR constructs versus the endogenous CBP protein. The cells were treated with the indicated inhibitor at a 1 μM concentration for 1 hour. Then, the stated concentration of dCBP-1 PROTAC was added for 3 hours, in addition to the previous concentration of the same inhibitor. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

Article Snippet: The CBP/p300 bromodomain inhibitor: GNE-049 (MedChemExpress, HY-108435), CBP/p300 PROTAC: dCBP-1 (MedChemExpress, HY-134582), BRD4 bromodomain inhibitor: (+)-JQ-1 (MedChemExpress, HY-13030), BRD4 PROTAC: ARV-825 (MedChemExpress, HY-16954), BRD9 bromodomain inhibitor: iBRD9 (MedChemExpress, HY-18975), and broad-spectrum bromodomain inhibitor: Bromosporine (MedChemExpress, HY-15815) were dissolved in DMSO and then diluted to 1μM in mESC media for 24-hour treatments, unless stated otherwise.

Techniques: Binding Assay, Fluorescence, Flow Cytometry, Concentration Assay, Construct

Journal: Molecular Oncology

Article Title: FET fusion oncoproteins interact with BRD4 and SWI/SNF chromatin remodelling complex subtypes in sarcoma

doi: 10.1002/1878-0261.13195

Figure Lengend Snippet: Composition of FET‐FOP‐bound SWI/SNF complexes.

Article Snippet: The BRD4 PROTAC degrader ARV‐825 (S8297, Selleckchem, Houston, TX, USA) was dissolved in DMSO, aliquoted in stock solutions of 100 m m and stored at −80 °C.

Techniques: Tandem Mass Spectroscopy

Journal: Molecular Oncology

Article Title: FET fusion oncoproteins interact with BRD4 and SWI/SNF chromatin remodelling complex subtypes in sarcoma

doi: 10.1002/1878-0261.13195

Figure Lengend Snippet: BRD4 expression, interactions and inhibition. (A) Schematic visualization of BRD4 long isoform (BRD4‐L) and short (BRD4‐S). Amino acid numbers and potential SUMO post‐translational modification sites (PTM) are indicated. (B) Western blot analysis (WB) of BRD4 isoforms in 10 µg nuclear extracts (extracted in 500 m m KCl) of MLS 402‐91, 2645‐94 and 1765‐92, EWS TC‐71 and HT1080 fibrosarcoma, using a BRD4 antibody (ab128874, N‐term, both isoforms). Analysis with two more BRD4 antibodies (C‐term) are shown in Fig. . (C) Western blot analysis of MLS 1765‐92, 402‐91 and 2645‐94, and EWS TC‐71 SSE extracts visualizing short and long BRD4 isoforms (BRD4 antibody ab128874). Same samples and loading as in Fig. . (D) Quantitative western blot analysis of BRG1‐biotin immunoprecipitated (IP) sequential salt extracts (250, 500 and 1000 m m KCl) in MLS 402‐91 and EWS TC‐71, visualizing BRD4 isoform co‐IP (BRD4 antibody ab128874). Same samples and loading as in Fig. . (E) Western blot analysis of BRG1‐biotin immunoprecipitated nuclear extracts of HT1080 fibrosarcoma cells transiently transfected (24h) with FUS‐DDIT3‐EGFP, EWSR1‐FLI1‐EGFP or EGFP control (R1) visualizing successful co‐IP of the SWI/SNF complex (BAF57) and BRD4. R2‐R3 are displayed in Fig. S4E. (F) Cell viability dose response curves of MLS cell lines 2645‐94, 402‐91 and 1765‐92, EWS TC‐71, HT1080 fibrosarcoma and fibroblasts (F470) after BRD4 inhibition (AZD5153, 72h). Mean +/‐ SD (standard deviation) is shown, n = 12 (2 biological, 6 technical replicates each). (G) Western blot analysis of BRG1‐biotin immunoprecipitated nuclear extracts of MLS 402‐91 control and BRD4‐inhibited cells (BRD4i, AZD5153, 24 h 500 n m ) visualizing SWI/SNF components (BRG1, BAF57 and BAF47), FUS‐DDIT3, normal FET protein EWSR1 and BRD4. (E, G) Loading IP‐WB: Maximum amount of eluate (B) and non‐bound (NB) were loaded on the gel. Input (I) samples were diluted relative NB and around 5% of input was loaded.

Article Snippet: The BRD4 PROTAC degrader ARV‐825 (S8297, Selleckchem, Houston, TX, USA) was dissolved in DMSO, aliquoted in stock solutions of 100 m m and stored at −80 °C.

Techniques: Expressing, Inhibition, Modification, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Control, Standard Deviation

Journal: Molecular Oncology

Article Title: FET fusion oncoproteins interact with BRD4 and SWI/SNF chromatin remodelling complex subtypes in sarcoma

doi: 10.1002/1878-0261.13195

Figure Lengend Snippet: ChIP sequencing reveals co‐localization of FUS‐DDIT3, SWI/SNF components and BRD4. (A) ChIP‐seq peak profiles of BAF155, BRG1 and FUS‐DDIT3 in MLS 402‐91 ± 3 kb surrounding TSS (transcription start site). (B) Bar chart showing the genomic distribution of BAF155, BRG1 and FUS‐DDIT3 ChIP‐seq peaks in MLS 402‐91. The majority of peaks are located in promotors close to the TSS, in introns or distal intergenic sites. UTR: mRNA untranslated region. (C) Venn diagram depicting overlap of BAF155, BRG1 and FUS‐DDIT3 ChIP binding sites and annotated genes in MLS 402‐91. The same gene may appear multiple times. The top de novo motif identified with Homer motif discovery for the combined binding sites is shown. (D) Significantly enriched gene sets from the “Reactome” gene set collection using the 4461 unique genes bound by FUS‐DDIT3 and at least one SWI/SNF component. Top 10 based on gene ratio is shown. Gene count is indicated by dot size and P (adjusted)‐value by colour. (E) Venn diagram depicting overlap of genes significantly regulated by ectopic FUS‐DDIT3‐EGFP expression (adjusted P ‐value ≤ 0.05 and Log2 fold change≥ 1) and unique genes bound by FUS‐DDIT3 and at least one SWI/SNF component. Significant enrichment was determined by Fisher’s exact test ( P < 1e‐10). (F) Venn diagram depicting overlap of BAF155, BRG1 and FUS‐DDIT3 ChIP binding sites in MLS 402‐91 from our dataset combined with BRD4 binding sites from Chen et al. dataset.

Article Snippet: The BRD4 PROTAC degrader ARV‐825 (S8297, Selleckchem, Houston, TX, USA) was dissolved in DMSO, aliquoted in stock solutions of 100 m m and stored at −80 °C.

Techniques: ChIP-sequencing, Binding Assay, Expressing

Journal: Molecular Oncology

Article Title: FET fusion oncoproteins interact with BRD4 and SWI/SNF chromatin remodelling complex subtypes in sarcoma

doi: 10.1002/1878-0261.13195

Figure Lengend Snippet: The interactomes of FET oncoproteins are enriched in phase separation propensity and transcriptional components. (A) Significantly enriched Panther protein class for FUS‐DDIT3‐interacting proteins. Percentage of proteins in protein class versus total of proteins matched to a protein class. (B) Significantly enriched gene sets from the “Reactome” and “Gene ontology (GO) biological processes” gene set collections for FUS‐DDIT3‐interacting proteins. Top 10 based on gene ratio is shown. Gene count is indicated by dot size and q‐value by colour. (C) Pie charts of FUS‐DDIT3 and EWSR1‐FLI1‐interacting proteins with phase separation propensity score (PScore) above or below the cutoff at 4. (D) Visualization of phase separation propensity score (PScore) of FET oncoproteins and their parental proteins. Pie chart shows proteins above or below the cutoff at 4. Blue dot indicates proteins with a high phase separation propensity, above the cutoff, visualized by black line. (E) Visualization of phase separation propensity score (PScore) of SWI/SNF components. Pie chart shows proteins above or below the cutoff at 4. Red dot indicates proteins with a high phase separation propensity, above the cutoff, visualized by black line. (F) Schematic visualization of potential BRD4, mediator, RNA polymerase II and FET‐FOP‐bound SWI/SNF complex interactions near chromatin. (G) Immunofluorescence staining of HT1080 cells transiently transfected with FUS‐DDIT3‐EGFP, probed with BRG1/BRD4, BRG1/MED1 and FUS‐DDIT3‐EGFP/BRD4 and analyzed with laser scanning microscopy. Representative images are shown. Scale bar: 5 µm. (H) Pearson’s correlation coefficient for co‐localization of BRG1 vs. BRD4, BRG1 vs. MED1, FUS‐DDIT3 vs. BRD4, FUS‐DDIT3 vs. BRG1 and FUS‐DDIT3 vs. MED1 in HT1080 cells transiently transfected with FUS‐DDIT3‐EGFP. (I) Manders’ split coefficients for co‐localization of BRG1 vs. BRD4, BRG1 vs. MED1, FUS‐DDIT3 vs. BRD4, FUS‐DDIT3 vs. BRG1 and FUS‐DDIT3 vs. MED1 in HT1080 cells transiently transfected with FUS‐DDIT3‐EGFP. The dark grey bar corresponds to the fraction of protein 1 (e.g. BRG1) overlapping with protein 2 (e.g. BRD4) and the light grey corresponds to the fraction of protein 2 (e.g. BRD4) overlapping with protein 1 (e.g. BRG1).

Article Snippet: The BRD4 PROTAC degrader ARV‐825 (S8297, Selleckchem, Houston, TX, USA) was dissolved in DMSO, aliquoted in stock solutions of 100 m m and stored at −80 °C.

Techniques: Immunofluorescence, Staining, Transfection, Laser-Scanning Microscopy