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 how the Parbit expression cassette is used to generate stably expressed Acyl-eCRs in mESCs. RMCE by Cre, followed by a double selection of ganciclovir and puromycin, was applied to generate these constructs at a defined site in the mouse genome. A CAG promoter drives the constitutive expression of the bromodomain of interest, which is fused to a nuclear localization signal (NLS), and an eGFP tag. This construct also fuses a biotin acceptor site to the N-terminus of the protein, which can be biotinylated in vivo by a bacterial BirA ligase. (B) Schematic diagram showing how CBP was endogenously tagged with an eGFP tag. A homology donor construct was generated by cloning a 900 bp upstream and a 1,048 bp (CBP) or 1,068 bp (p300) downstream homology arm flanking a 30 bp flexible GGS linker that was fused to an eGFP tag. This donor construct was co-transfected with a pX330 CRISPR-Cas9 plasmid, which had an sgRNA targeting the C-terminus of the CBP gene. (C-D) Sanger sequencing of genotyping PCR products from the C-terminus of the CBP (C) and p300 (D) loci, confirming the in-frame homologous integration of an eGFP tag. Data shown are from mESC clone #1 for both CBP and p300 tagging. (E) Flow cytometry data showing the eGFP signal from cell lines where either CBP or p300 were endogenously tagged with eGFP. Two clonal replicates for each protein tagging are shown. Cell lines were maintained in culture for more than two weeks to demonstrate stable expression of the eGFP fusion proteins. (F) Western blot of nuclear extracts from cell lines treated with 1 μM dCBP-1 PROTAC for the stated duration. An antibody against GFP was used to probe the eGFP tag on the Acyl-eCR constructs. The CBP_BRD.1x eCR runs at approximately 60 kDa, and the Empty-eGFP construct runs at 33 kDa. Non-specific bands are marked by an asterisk (*). Revert700 Total Protein Stain is used to show equal loading in lanes.

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: Expressing, Stable Transfection, Selection, Construct, In Vivo, Generated, Cloning, Transfection, CRISPR, Plasmid Preparation, Sequencing, Flow Cytometry, Western Blot, Staining

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: 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) Phylogenetic tree showing that the panel of Acyl-eCRs comprises representative bromodomains from all classes of bromodomains. All bromodomain protein sequences were obtained from InterPro and aligned with Clustal Omega’s multiple sequence alignment. Bromodomains are classified based on structure & druggability . Black circles represent the bromodomains that are included in the panel of Acyl-eCR cell lines. (B) Normalized FACS data showing the effects of dCBP-1 PROTAC treatments on all Acyl-eCR cell lines. PROTAC was added at a 1 μM concentration for 24 hours of treatment. 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: Sequencing, Concentration Assay

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 how nuclei isolation & permeabilization followed by flow cytometry can measure the retention of proteins on chromatin in lieu of high background fluorescence. Nuclei can be harvested and permeabilized from whole cells, and then washed to remove the unbound or weakly bound fraction of the protein of interest. Since the weakly bound fraction of protein is removed, the fraction of protein remaining can be measured at a better signal-to-noise ratio via flow cytometry. (B) Flow cytometry analysis of nuclei harvested from BRD9_BRD.1x or WT cells. N3 gate shows the GFP signal being measured in nuclei, after iBRD9 or control treatments. Treatments in the WT cell line show a change in autofluorescence in the nuclei from the drug treatments. (C) Normalized flow cytometry data showing how Acyl-eCRs with 1x or 2x copies of the BRD9 bromodomain remain bound to chromatin, after iBRD9 treatments. iBRD9 treatments were performed at 1 μM concentration for 24 hours. 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: Isolation, Flow Cytometry, Fluorescence, Control, Concentration Assay

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 how nuclei isolation & permeabilization followed by flow cytometry can measure the retention of proteins on chromatin in lieu of high background fluorescence. Nuclei can be harvested and permeabilized from whole cells, and then washed to remove the unbound or weakly bound fraction of the protein of interest. Since the weakly bound fraction of protein is removed, the fraction of protein remaining can be measured at a better signal-to-noise ratio via flow cytometry. (B) Flow cytometry analysis of nuclei harvested from BRD9_BRD.1x or WT cells. N3 gate shows the GFP signal being measured in nuclei, after iBRD9 or control treatments. Treatments in the WT cell line show a change in autofluorescence in the nuclei from the drug treatments. (C) Normalized flow cytometry data showing how Acyl-eCRs with 1x or 2x copies of the BRD9 bromodomain remain bound to chromatin, after iBRD9 treatments. iBRD9 treatments were performed at 1 μM concentration for 24 hours. 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: Isolation, Flow Cytometry, Fluorescence, Control, Concentration Assay

Journal: Science Advances

Article Title: BRD9 functions as a methylarginine reader to regulate AKT-EZH2 signaling

doi: 10.1126/sciadv.ads6385

Figure Lengend Snippet: ( A ) Schematic of peptide pull-down and mass spectrometry experiment using biotin-labeled AKT1-R391 peptides with or without symmetrical dimethylarginine (R391me2s and R391me0). ( B ) A scatterplot comparing the log 2 iBAQ protein intensities between the R391me2s and R391me0. BRD9 was marked as red. n = 1 biological replicate. ( C ) Immunoblot (IB) analysis of pull-down products by biotin-labeled AKT1 peptides and myc-BRD9. ( D ) Schematic showing the domains of BRD9. ( E and F ) IB analysis of WCL and immunoprecipitates (IPs) derived from HEK293T cells transfected with the indicated constructs. EV, empty vector. ( G ) IB analysis of pull-down products derived from biotin-labeled peptides and BRD9-BD or BRD4-BD1 purified from bacteria. ( H ) Zoomed-out (left) and zoomed-in (right) images depicting the proposed interaction of R391-me2s (blue) from AKT (orange) with BRD9-BD (gray) based on MD simulations. Hydrogen bonds (yellow) and key residues on BRD9 (purple) were shown. ( I ) IB analysis of WCL and hemagglutinin (HA)–IP derived from HEK293T cells transfected with Myc-BRD9-WT or mutants and HA-AKT1 constructs.

Article Snippet: I-BRD9 (HY-18975), GSK126 (HY-13470), ipatasertib (HY-15186), LY294002 (HY-10108), BKM120 (HY-70063), and AU-15330 (HY-145388) were purchased from MedChemExpress. dBRD9-A (6943) was purchased from TOCRIS.

Techniques: Mass Spectrometry, Labeling, Western Blot, Derivative Assay, Transfection, Construct, Plasmid Preparation, Purification, Bacteria

Journal: Science Advances

Article Title: BRD9 functions as a methylarginine reader to regulate AKT-EZH2 signaling

doi: 10.1126/sciadv.ads6385

Figure Lengend Snippet: ( A ) IB analysis of WCL derived from MDA-MB-231 cells depleted of BRD9 by two independent sgRNAs. sgGFP is a negative control. ( B ) IB analysis of WCL derived for MDA-MB-231 cells treated with 100 nM dBRD9 for the indicated time. ( C ) IB analysis of WCL derived from control (sgGFP) or BRD9-depleted MDA-MB-231 cells. Cells were serum starved for 16 hours before being treated with 100 nM insulin for 1 hour. ( D ) IB analysis of WCL derived from BRD9-depleted MDA-MB-231 cells stably expressing HA-BRD9-WT or HA-BRD9-3Mut. ( E ) IB analysis of WCL derived for MDA-MB-231 cells treated with 5 μM I-BRD9 for the indicated time. ( F ) IB analysis of WCL and HA-IPs derived from HEK293T cells transfected with indicated constructs. ( G ) IB analysis of WCL and GST pull-downs derived from HEK293T cells transfected with indicated constructs. ( H ) Schematic model depicting that BRD9 recognizes AKT1-R391me2s to impair the interaction between PH and KD domains, leading to AKT membrane translocation and activation.

Article Snippet: I-BRD9 (HY-18975), GSK126 (HY-13470), ipatasertib (HY-15186), LY294002 (HY-10108), BKM120 (HY-70063), and AU-15330 (HY-145388) were purchased from MedChemExpress. dBRD9-A (6943) was purchased from TOCRIS.

Techniques: Derivative Assay, Negative Control, Control, Stable Transfection, Expressing, Transfection, Construct, Membrane, Translocation Assay, Activation Assay

Journal: Science Advances

Article Title: BRD9 functions as a methylarginine reader to regulate AKT-EZH2 signaling

doi: 10.1126/sciadv.ads6385

Figure Lengend Snippet: ( A ) Analysis of BRD9 mRNA levels in breast tumors and normal breast tissues in the TCGA dataset. ** P < 0.01, *** P < 0.001, one-way ANOVA. ( B ) KEGG pathway enrichment analysis of DEGs between BRD9-high and BRD9-low breast cancer samples in the TCGA dataset. ( C ) Gene Set Enrichment Analysis (GSEA) of the PI3K-AKT signaling pathway in BRD9-high and BRD9-low breast cancer samples in the TCGA dataset. NES, normalized enrichment score. ( D ) Control (sgGFP) or BRD9-depleted MDA-MB-231 cells were subjected to xenograft mouse assays. Tumor size was measured every other day. Data are shown as means ± SEM of n = 8 tumors for each group. *** P < 0.001, two-way ANOVA. ( E ) Dissected tumors were weighed. Data are shown as the means ± SEM of n = 8 tumors for each group. * P < 0.05, Student’s t test. ( F ) IB analysis of lysates derived from tumor tissues in (E). ( G ) MDA-MB-231 cells expressing BRD9-WT or BRD9-3Mut were subjected to xenograft mouse assays. Tumor size was measured every 3 days. Data are shown as means ± SEM of n = 8 tumors for each group. *** P < 0.001, two-way ANOVA. ( H ) Dissected tumors were weighed. Data are shown as the means ± SEM of n = 8 tumors for each group. ** P < 0.01, Student’s t test. ( I ) IB analysis of lysates derived from tumor tissues in (H).

Article Snippet: I-BRD9 (HY-18975), GSK126 (HY-13470), ipatasertib (HY-15186), LY294002 (HY-10108), BKM120 (HY-70063), and AU-15330 (HY-145388) were purchased from MedChemExpress. dBRD9-A (6943) was purchased from TOCRIS.

Techniques: Control, Derivative Assay, Expressing

Journal: Science Advances

Article Title: BRD9 functions as a methylarginine reader to regulate AKT-EZH2 signaling

doi: 10.1126/sciadv.ads6385

Figure Lengend Snippet: ( A ) Venn diagrams depicting common significant down-regulated and up-regulated genes ( Q value < 0.05) between I-BRD9 and ipatasertib (AKTi). MDA-MB-231cells were treated with 5 μM I-BRD9 or 1 μM ipatasertib for 16 hours prior to determination of gene expression by RNA-seq. DMSO treatment as a negative control. ( B ) Heatmap showing mRNA expression changes of commonly regulated genes described in (A). ( C ) Identification of hallmark signaling pathways shared by I-BRD9 and ipatasertib. ( D ) Boxplots of common down-regulated genes based on the number of H3K27me3 mark analyzed using epidecodeR software. “0” represents no H3K27me3 mark, and “1+” represents ≥1 H3K27me3 mark. ( E ) RT-qPCR analysis of mRNA levels of select genes commonly regulated by I-BRD9 and ipatasertib. MDA-MB-231 cells were treated with 5 μM I-BRD9 or 1 μM ipatasertib for 16 hours. Data are shown as means ± SD of n = 3 biological replicates. ** P < 0.01 and *** P < 0.001, one-way ANOVA and Tukey post hoc test. ( F ) RT-qPCR analysis of mRNA levels of select common genes in MDA-MB-231 cells expressing BRD9-WT or BRD9-3Mut. Data are shown as means ± SD of n = 3 biological replicates. * P < 0.05 and ** P < 0.01, Student’s t test.

Article Snippet: I-BRD9 (HY-18975), GSK126 (HY-13470), ipatasertib (HY-15186), LY294002 (HY-10108), BKM120 (HY-70063), and AU-15330 (HY-145388) were purchased from MedChemExpress. dBRD9-A (6943) was purchased from TOCRIS.

Techniques: Gene Expression, RNA Sequencing, Negative Control, Expressing, Software, Quantitative RT-PCR

Journal: Science Advances

Article Title: BRD9 functions as a methylarginine reader to regulate AKT-EZH2 signaling

doi: 10.1126/sciadv.ads6385

Figure Lengend Snippet: ( A ) IB analysis of WCL derived from MDA-MB-231 cells infected with lentivirus of sgGFP or sgBRD9. ( B ) IB analysis of WCL derived from MDA-MB-231 cells treated with indicated doses of dBRD9 for 16 hours. ( C ) IB analysis of WCL derived from MDA-MB-231 cells infected with lentivirus of sgGFP or sgBRD9. Cells were treated with 5 μM GSK126 for 16 hours before harvesting. ( D ) IB analysis of WCL derived from MDA-MB-231 cells treated with indicated doses of I-BRD9 for 16 hours. ( E ) IB analysis of WCL derived from MDA-MB-231 cells stably expressing EV or Myr-AKT1 and depleted of BRD9 infected with lentivirus sgGFP (−) or sgBRD9 (+). ( F ) IB analysis of WCL derived from control (sgGFP) or AKT1-depleted MDA-MB-231 cells. Cells were treated with 5 μM I-BRD9 for 16 hours before harvesting. ( G ) RT-qPCR analysis of mRNA levels of select genes in MDA-MB-231 cells treated with DMSO or 5 μM GSK126 for 48 hours. Data are shown as means ± SD of n = 3 biological replicates. * P < 0.05, ** P < 0.01, and *** P < 0.001, Student’s t test.

Article Snippet: I-BRD9 (HY-18975), GSK126 (HY-13470), ipatasertib (HY-15186), LY294002 (HY-10108), BKM120 (HY-70063), and AU-15330 (HY-145388) were purchased from MedChemExpress. dBRD9-A (6943) was purchased from TOCRIS.

Techniques: Derivative Assay, Infection, Stable Transfection, Expressing, Control, Quantitative RT-PCR

Journal: Science Advances

Article Title: BRD9 functions as a methylarginine reader to regulate AKT-EZH2 signaling

doi: 10.1126/sciadv.ads6385

Figure Lengend Snippet: ( A ) Inhibition of cell viability and dose-response matrixes analyzed by SynergyFinder. MDA-MB-231 cells were treated with the indicated doses of I-BRD9 and GSK126 for 96 hours prior to analysis of cell viability. ( B ) MDA-MB-231 cells treated with I-BRD9 or GSK126 were subjected to cell proliferation assays. Data are shown as means ± SD of n = 3 biological replicates. *** P < 0.001, two-way ANOVA and Tukey post hoc test. ( C ) MDA-MB-231 cells treated with I-BRD9 or GSK126 were subjected to colony formation assays. Representative images are shown. ( D ) Quantification of colonies in (C). Data are shown as means ± SD of n = 3 biological replicates. * P < 0.05 and *** P < 0.001, one-way ANOVA and Tukey post hoc test. ( E ) Schematic of a mouse xenograft assay to evaluate the antitumor effects of I-BRD9 and GSK126. ( F ) Tumor growth curve upon treatment of I-BRD9 and GSK126. Data are shown as means ± SEM of n = 6 mice for each group. * P < 0.05, two-way ANOVA and Tukey post hoc test. ( G and H ) Dissected tumors were weighed. Data are shown as the means ± SEM of n = 6 tumors for each group. * P < 0.05, one-way ANOVA and Tukey post hoc test. ( I ) Representative images of TUNEL assays in xenograft tumors in (G). ( J ) Schematic depicting the function of the BRD9-AKT-EZH2 axis in regulating transcription and tumor growth.

Article Snippet: I-BRD9 (HY-18975), GSK126 (HY-13470), ipatasertib (HY-15186), LY294002 (HY-10108), BKM120 (HY-70063), and AU-15330 (HY-145388) were purchased from MedChemExpress. dBRD9-A (6943) was purchased from TOCRIS.

Techniques: Inhibition, Xenograft Assay, TUNEL Assay

Journal: Cell death & disease

Article Title: Prostate cancer exploits BRD9-driven metabolic reprogramming to shape the aggressive phenotype.

doi: 10.1038/s41419-025-07561-9

Figure Lengend Snippet: Fig. 2 BRD9 affects REDOX balance in PCa cells. A DEGs in RNA-seq from LNCaP-AI shBRD9 cells were subjected to GO enrichment analysis. B Functional enrichment of BRD9 target genes in C4-2B cells from ChIP analysis. C Relative expression levels of HK2, PKM, LDHA, and G6PD in LNCaP cells transfected with siBRD9 or siNC 24 h with or without androgen deprivation (n = 3). D–F Levels of glucose consumption, lactate production, and G6PD activity in LNCaP cells (n = 3). G–I NADPH/NADP+ ratio, GSH/GSSG ratio, and ROS level in LNCaP cells transfected with BRD9 overexpression for 24 h with or without androgen deprivation. J–L NADPH/NADP+ ratio, GSH/GSSG ratio, and ROS level in C4-2B cells transfected with siBRD9 or siNC for 24 h (n = 3). M mRNA levels of antioxidant-related molecules in C4-2B cells (n = 3). N, O Membrane potential in C4-2B and LNCaP cells with BRD9 knockdown or overexpression with or without androgen deprivation (n = 3). P, Q Colony formation and CCK-8 assays in C4-2B cells transfected with siNC or siBRD9 in the presence of ROS scavenger NAC (5 nM) (n = 3). Representative images are shown in the left panel and quantitative analysis is shown in the right panel (P). Two-tailed unpaired t-test, one-way and two-way analysis of variance (ANOVA). Error bars represent SD; *P < 0.05, **P < 0.01, *** P < 0.001.

Article Snippet: BRD9 inhibitor (I-BRD9) was purchased from Selleck.

Techniques: RNA Sequencing, Functional Assay, Expressing, Transfection, Activity Assay, Over Expression, Membrane, Knockdown, CCK-8 Assay, Two Tailed Test

Journal: Cell death & disease

Article Title: Prostate cancer exploits BRD9-driven metabolic reprogramming to shape the aggressive phenotype.

doi: 10.1038/s41419-025-07561-9

Figure Lengend Snippet: Fig. 3 BRD9 promotes PCa cell growth through PYGL in PCa cells. A, B The mRNA and protein levels of PYGL in LNCaP cells with short- period androgen-deprivation treatment (24, 48, 72, and 96 h) determined by qRT-PCR and western blotting (n = 3). FBS fatal bovine serum, CSS charcoal-stripped serum, h hours. C Expression levels of PYGL mRNA in LNCaP cells transfected with siBRD9 or siNC 24 h post with or without androgen deprivation plus NAC treatment determined by qRT-PCR analyses (n = 3). D Correlation between the relative levels of BRD9 and PYGL mRNA transcripts in PCa tissues from the GSE2443 database. E The prediction of the binding site of BRD9 on the PYGL promoter based on ChIP-seq analysis. F ChIP analysis of the BRD9 enrichment on the PYGL promoter in LNCaP cells. Results show the relative enrichment with the anti-BRD9 antibody versus the IgG control. G, H EdU and colony formation assays in C4-2B cells with transfection of siBRD9 for 24 h and then the PYGL overexpression plasmid for another 24 h (n = 3). Scale bars, 20 μm. I, J EdU and colony formation assays in C4-2B cells with transfection of siPYGL for 24 h and then the BRD9 overexpression plasmid for another 24 h (n = 3). Scale bars, 20 μm. Spearman’s rank test, one-way and two-way analysis of variance (ANOVA). Error bars represent SD; *P < 0.05, **P < 0.01, *** P < 0.001.

Article Snippet: BRD9 inhibitor (I-BRD9) was purchased from Selleck.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Transfection, Binding Assay, ChIP-sequencing, Control, Over Expression, Plasmid Preparation

Journal: Cell death & disease

Article Title: Prostate cancer exploits BRD9-driven metabolic reprogramming to shape the aggressive phenotype.

doi: 10.1038/s41419-025-07561-9

Figure Lengend Snippet: Fig. 4 BRD9 maintains REDOX balance via PYGL in PCa cells. A–C Glucose consumption, lactate production, and G6PD activity in C4-2B cells with transfection of siBRD9 for 24 h and then the PYGL overexpression plasmid for another 24 h (n = 3). D–F NADPH/NADP+ ratio, GSH/GSSG ratio, and ROS level in LNCaP cells with PYGL knockdown with or without androgen deprivation (n = 3). FBS fatal bovine serum, CSS charcoal- stripped serum. G–I NADPH/NADP+ ratio, GSH/GSSG ratio, and ROS level in C4-2B cells with transfection of siBRD9 for 24 h and then the PYGL overexpression plasmid for another 24 h (n = 3). J, K OCR in C4-2B cells co-transfected with siBRD9 for 24 h and then the PYGL overexpression plasmid for another 24 h or co-transfected with siPYGL siRNA for 24 h and then the BRD9 overexpression plasmid for another 24 h (n = 3). Representative recordings of OCR during extracellular flow analysis (“Seahorse”) are shown in the up panel, and quantitative analysis of the calculated basal and maximum respiratory rates, ATP production rate, and spare respiratory capacity are shown in the bottom panel. L, M Membrane potential in C4-2B cells co-transfected with siBRD9 for 24 h and then the PYGL overexpression plasmid for another 24 h or co-transfected with siPYGL siRNA for 24 h and then the BRD9 overexpression plasmid for another 24 h (n = 3). One-way and two-way analysis of variance (ANOVA). Error bars represent SD; *P < 0.05, **P < 0.01, *** P < 0.001.

Article Snippet: BRD9 inhibitor (I-BRD9) was purchased from Selleck.

Techniques: Activity Assay, Transfection, Over Expression, Plasmid Preparation, Knockdown, Membrane

Journal: Cell death & disease

Article Title: Prostate cancer exploits BRD9-driven metabolic reprogramming to shape the aggressive phenotype.

doi: 10.1038/s41419-025-07561-9

Figure Lengend Snippet: Fig. 6 BRD9 maintains REDOX balance and PPP activity in PCa cells through NFYA. A KEGG pathway gene set enrichment analysis of the potential BRD9 interactors identified by co-immunoprecipitation assays and mass spectrometry. B Kaplan-Meier survival analysis of PCa cases from the GEPIA prostate cohort according to relative expression of NFYA. C Correlation between the relative levels of BRD9 and NFYA mRNA transcripts in prostate cancer tissues of the GEPIA database. D Localization of BRD9 (red) and NFYA (green) in LNCaP cell nuclei was verified by immunofluorescent staining with or without androgen deprivation. Magnified images from the regions marked by rectangles in the top panel were showed in the bottom panel (n = 3). Scale bars, 2 μm. E Binding potential between BRD9 and NFYA or BRG1 in the whole cell lysates was determined by co-immunoprecipitation assays in LNCaP cells with or without androgen deprivation (n = 3). IgG served as negative control. ChIPanalysis using LNCaPor C4-2B cells to validate (F) NFYA or (G) BRD9 enrichment on the PYGL promoter (n = 3 for each panel). H Relative levels of PYGL mRNA expression in C4-2B cells transfected with empty vector or the BRD9 overexpression plasmid with or without NFYA suppression (n = 3). I–N Glucose consumption, lactate production, G6PD activity, NADPH/NADP+ ratio, GSH/GSSG ratio, and ROS level in C4-2B cells transfected with empty vector or BRD9 overexpression plasmid with or without NFYA suppression (n = 3). O Relative levels of PYGL mRNA expression in LNCaP cells transfected with wild-type Flag-BRD9 vector (WT), or vectors expressing Flag-BRD9 with single or double cysteine mutations with or without androgen deprivation (n = 3). FBS fatal bovine serum, CSS charcoal-stripped serum. Kaplan–Meier survival analysis, Spearman’s rank test, one-way and two-way analysis of variance (ANOVA). Error bars represent SD; *P < 0.05, **P < 0.01, *** P < 0.001.

Article Snippet: BRD9 inhibitor (I-BRD9) was purchased from Selleck.

Techniques: Activity Assay, Immunoprecipitation, Mass Spectrometry, Expressing, Staining, Binding Assay, Negative Control, Transfection, Plasmid Preparation, Over Expression