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TargetMol brd9 inhibitor
Brd9 Inhibitor, supplied by TargetMol, used in various techniques. Bioz Stars score: 96/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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brd9 inhibitor - by Bioz Stars, 2026-03

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(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.

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

(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.

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.

Techniques: Immunofluorescence, Fluorescence, Expressing

(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.

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.

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

(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.

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.

Techniques: Sequencing, Concentration Assay

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

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.

Techniques: Isolation, Flow Cytometry, Fluorescence, Control, Concentration Assay

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.

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

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.

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

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.

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

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.

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

A ASE categories, including skipped exon (SE), alternative 3′ splice site usage (A3SS), alternative 5′ splice site usage (A5SS), mutually exclusive exon usage (MXE), and intron retention (RI). B Bar plot displaying numbers of all identified ASEs in the comparison of 18 SF3B1 MUT and 17 SF3B1 WT subset #2 cases across different ASE categories. 163,097 ASEs were detected and involved transcripts of 10,437 genes. C Volcano plot depicting all identified ASEs in the comparison of SF3B1 MUT and SF3B1 WT subset #2 cases. Red dots indicate ASEs that are considered significant (|ΔPSI | ≥ 20% and FDR ≤ 0.01; 80 ASEs). D Bar plot displaying numbers of significant ASEs in the comparison of SF3B1 MUT and SF3B1 WT subset #2 cases across different ASE categories. 80 ASEs were considered significant and involved transcripts of 62 genes. E Heatmap illustrating the individual PSI values for the 80 significant ASEs detected in the comparison of SF3B1 MUT and SF3B1 WT subset #2 cases. 33 ASEs occurred within the same gene at least twice. ASEs are displayed based on the ASE category. The bar plot to the right of the heatmap shows ΔPSI values. The gene affected by each ASE and the corresponding unique ASE ID are listed. Genes that encode ncBAF complex-interacting proteins are depicted in color. For multiple ASEs per gene, only the top ASE is colored, while the others are marked with black asterisks. Additional details of these ASEs are provided in Supplementary Table . F Model of the ncBAF chromatin remodeling complex and previously reported ncBAF complex interactors. Alternatively spliced transcripts identified in SF3B1 MUT subset #2 CLL that encode ncBAF complex-related proteins are depicted in color. G Scattered box plot showing the PSI value distribution, ΔPSI values, and P values (Wilcoxon rank-sum test) for significant ncBAF complex-related ASEs identified in the comparison of SF3B1 MUT and SF3B1 WT subset #2 cases. The specific ASEs for ZEB1 , BRD9 , PLSCR1 , TENT4B , CXXC1 , DCAF16 , UBP1 , DLST , and SERBP1 have the unique ASE IDs ZEB1_SE_27141, BRD9_SE_53886, PLSCR1_SE_92684, PAPD5_SE_94061, CXXC1_SE_102397, DCAF16_SE_4627, UBP1_SE_85897, DLST_A3SS_2409, and SERBP1_A3SS_1321, respectively, in Supplementary Table . The boxes represent the median and the interquartile range (IQR), while the whiskers extend to 1.5 times the IQR from the first and third quartiles. ASE: alternative splicing event; WT: wildtype; MUT: mutated; FDR: false discovery rate; PSI: percent spliced in; VAF: variant allele frequency.

Journal: Leukemia

Article Title: The non-canonical BAF chromatin remodeling complex is a novel target of spliceosome dysregulation in SF3B1 -mutated chronic lymphocytic leukemia

doi: 10.1038/s41375-024-02379-4

Figure Lengend Snippet: A ASE categories, including skipped exon (SE), alternative 3′ splice site usage (A3SS), alternative 5′ splice site usage (A5SS), mutually exclusive exon usage (MXE), and intron retention (RI). B Bar plot displaying numbers of all identified ASEs in the comparison of 18 SF3B1 MUT and 17 SF3B1 WT subset #2 cases across different ASE categories. 163,097 ASEs were detected and involved transcripts of 10,437 genes. C Volcano plot depicting all identified ASEs in the comparison of SF3B1 MUT and SF3B1 WT subset #2 cases. Red dots indicate ASEs that are considered significant (|ΔPSI | ≥ 20% and FDR ≤ 0.01; 80 ASEs). D Bar plot displaying numbers of significant ASEs in the comparison of SF3B1 MUT and SF3B1 WT subset #2 cases across different ASE categories. 80 ASEs were considered significant and involved transcripts of 62 genes. E Heatmap illustrating the individual PSI values for the 80 significant ASEs detected in the comparison of SF3B1 MUT and SF3B1 WT subset #2 cases. 33 ASEs occurred within the same gene at least twice. ASEs are displayed based on the ASE category. The bar plot to the right of the heatmap shows ΔPSI values. The gene affected by each ASE and the corresponding unique ASE ID are listed. Genes that encode ncBAF complex-interacting proteins are depicted in color. For multiple ASEs per gene, only the top ASE is colored, while the others are marked with black asterisks. Additional details of these ASEs are provided in Supplementary Table . F Model of the ncBAF chromatin remodeling complex and previously reported ncBAF complex interactors. Alternatively spliced transcripts identified in SF3B1 MUT subset #2 CLL that encode ncBAF complex-related proteins are depicted in color. G Scattered box plot showing the PSI value distribution, ΔPSI values, and P values (Wilcoxon rank-sum test) for significant ncBAF complex-related ASEs identified in the comparison of SF3B1 MUT and SF3B1 WT subset #2 cases. The specific ASEs for ZEB1 , BRD9 , PLSCR1 , TENT4B , CXXC1 , DCAF16 , UBP1 , DLST , and SERBP1 have the unique ASE IDs ZEB1_SE_27141, BRD9_SE_53886, PLSCR1_SE_92684, PAPD5_SE_94061, CXXC1_SE_102397, DCAF16_SE_4627, UBP1_SE_85897, DLST_A3SS_2409, and SERBP1_A3SS_1321, respectively, in Supplementary Table . The boxes represent the median and the interquartile range (IQR), while the whiskers extend to 1.5 times the IQR from the first and third quartiles. ASE: alternative splicing event; WT: wildtype; MUT: mutated; FDR: false discovery rate; PSI: percent spliced in; VAF: variant allele frequency.

Article Snippet: Cell lines and primary CLL cells were treated with BRD9 inhibitors/degraders, I-BRD9 (SML1534; Sigma-Aldrich), PROTAC BRD9 Degrader-1 (HY-103632; MedChemExpress, New Jersey, NJ, USA), and dBRD9 Hydrochloride (SML2911; Sigma-Aldrich) [ , ].

Techniques: Comparison, Alternative Splicing, Variant Assay

Sashimi plots illustrating the identified ASEs and alternative splicing patterns in four genes that encode ncBAF complex-related proteins in SF3B1 WT versus SF3B1 MUT CLL; ZEB1 and BRD9 exhibit the top two ASEs in the SE category, while DLST and SERBP1 showcase the top two ASEs in the A3SS category. Sashimi plots for other ncBAF complex-related genes, PLSCR1 , TENT4B , CXXC1 , DCAF16 , and UBP1 , are presented in Supplementary Fig. . For each gene, the top two sashimi plots within the gray box illustrate the predicted splice variants in SF3B1 WT versus SF3B1 MUT CLL. The colored arc highlights the primary ASE, while lighter arcs represent additional ASEs if present. The gene map indicates the relative location and order of the detected exons in relation to the sequencing results. For each corresponding gene, the lower two sashimi plots show the coverage and splice junction count data from the aligned long-read RNA-seq data from an SF3B1 WT case (RS24) and an SF3B1 MUT case (RS55), both belonging to subset #2 CLL. The direction of the genes is arranged from left to right. WT: wildtype; MUT: mutated; PSI: percent spliced in.

Journal: Leukemia

Article Title: The non-canonical BAF chromatin remodeling complex is a novel target of spliceosome dysregulation in SF3B1 -mutated chronic lymphocytic leukemia

doi: 10.1038/s41375-024-02379-4

Figure Lengend Snippet: Sashimi plots illustrating the identified ASEs and alternative splicing patterns in four genes that encode ncBAF complex-related proteins in SF3B1 WT versus SF3B1 MUT CLL; ZEB1 and BRD9 exhibit the top two ASEs in the SE category, while DLST and SERBP1 showcase the top two ASEs in the A3SS category. Sashimi plots for other ncBAF complex-related genes, PLSCR1 , TENT4B , CXXC1 , DCAF16 , and UBP1 , are presented in Supplementary Fig. . For each gene, the top two sashimi plots within the gray box illustrate the predicted splice variants in SF3B1 WT versus SF3B1 MUT CLL. The colored arc highlights the primary ASE, while lighter arcs represent additional ASEs if present. The gene map indicates the relative location and order of the detected exons in relation to the sequencing results. For each corresponding gene, the lower two sashimi plots show the coverage and splice junction count data from the aligned long-read RNA-seq data from an SF3B1 WT case (RS24) and an SF3B1 MUT case (RS55), both belonging to subset #2 CLL. The direction of the genes is arranged from left to right. WT: wildtype; MUT: mutated; PSI: percent spliced in.

Techniques: Alternative Splicing, Sequencing, RNA Sequencing

A Scheme illustrating lentiviral constructs for SF3B1 WT and SF3B1 K700E overexpression under the control of a DOX-inducible promoter. B Bar plots depicting expression levels of endogenous and exogenous SF3B1 upon overexpression of SF3B1 WT and SF3B1 K700E in MEC1 and PCL12 cell lines. The bar plots display the mean values of triplicates with error bars representing the standard deviation. C Scheme illustrating the identified ASE in BRD9 with the regular and alternative splicing pattern. The blue and red boxes illustrate constant and alternative exons, respectively. D Increased expression of the alternative BRD9 transcript upon overexpression of SF3B1 K700E as compared to SF3B1 WT in MEC1 and PCL12 cell lines. The checkerboard scheme at the top of the panel illustrates the experimental conditions. The bar plots display the expression levels of BRD9 transcripts in general (transcripts containing exon 14), the regular BRD9 transcript (transcripts containing exon 14 followed by exon 16), and the alternative BRD9 transcript (transcripts containing exon 14 followed by exon 15) as determined by qPCR. Results are normalized on GAPDH levels and expressed as fold difference between DOX+ and DOX− conditions with corresponding P values (Student’s t -test). The bar plots display the mean values of experimental triplicates, with error bars representing the standard deviation. E Agarose gel electrophoresis images of PCR products spanning from exon 14 to 16 and allowing for exon 15 inclusion show a higher abundance of alternative BRD9 transcript in SF3B1 K700E DOX+. For a complete gel image see Supplementary Fig. . F Assessment of BRD9 splice variants in SF3B1 WT and SF3B1 MUT CLL, AML, and UVM cell lines. MEC1, PGA1, and HG3 are SF3B1 WT CLL cell lines, CII an SF3B1 K666E CLL cell line, HNT34 an SF3B1 K700E AML cell line, and MEL202 an SF3B1 R625G UVM cell line. The bar plots display the expression levels of BRD9 transcripts in general, the regular BRD9 transcript, and the alternative BRD9 transcript as determined by qPCR. Results for BRD9 transcripts in general are normalized on GAPDH levels, whereas regular and alternative BRD9 transcript levels are normalized on levels of BRD9 transcripts in general and expressed as fold difference between MEC1 and other cell lines with corresponding P values (Student’s t -test). The bar plots display the mean values of experimental triplicates, with error bars representing the standard deviation. G Agarose gel electrophoresis image of PCR products spanning from exon 14 to 16 and allowing for exon 15 inclusion shows a higher abundance of alternative BRD9 transcript in SF3B1 MUT cell lines. For a complete gel image see Supplementary Fig. . H Sashimi plots from the SF3B1 WT CLL cell line HG3 and the SF3B1 MUT AML and UVM cell lines, HNT34 and MEL202, respectively, illustrating the alternatively spliced exon 15 in BRD9 . The plots for HG3 are based on direct long-read RNA-seq, and HNT34 (SRR8616208) and MEL202 (SRR12354765) on short-read RNA-seq. I BRD9 splice isoform expression in SF3B1 WT and SF3B1 MUT CLL cell lines determined by isoform-specific antibodies. An antibody specific to the C-terminus of the regular BRD9 isoform detected a single band in both SF3B1 WT and SF3B1 MUT cell lines. Conversely, an antibody targeting a constant epitope within the central part of BRD9, detected two bands in the SF3B1 MUT cell line CII and one band in the SF3B1 WT cell lines MEC1 and PGA1, corresponding to the regular higher molecular weight BRD9 isoform and the alternative lower molecular weight BRD9 isoform. For complete blot images see Supplementary Fig. . WT: wildtype; MUT: mutated; DOX: doxycycline.

Journal: Leukemia

Article Title: The non-canonical BAF chromatin remodeling complex is a novel target of spliceosome dysregulation in SF3B1 -mutated chronic lymphocytic leukemia

doi: 10.1038/s41375-024-02379-4

Figure Lengend Snippet: A Scheme illustrating lentiviral constructs for SF3B1 WT and SF3B1 K700E overexpression under the control of a DOX-inducible promoter. B Bar plots depicting expression levels of endogenous and exogenous SF3B1 upon overexpression of SF3B1 WT and SF3B1 K700E in MEC1 and PCL12 cell lines. The bar plots display the mean values of triplicates with error bars representing the standard deviation. C Scheme illustrating the identified ASE in BRD9 with the regular and alternative splicing pattern. The blue and red boxes illustrate constant and alternative exons, respectively. D Increased expression of the alternative BRD9 transcript upon overexpression of SF3B1 K700E as compared to SF3B1 WT in MEC1 and PCL12 cell lines. The checkerboard scheme at the top of the panel illustrates the experimental conditions. The bar plots display the expression levels of BRD9 transcripts in general (transcripts containing exon 14), the regular BRD9 transcript (transcripts containing exon 14 followed by exon 16), and the alternative BRD9 transcript (transcripts containing exon 14 followed by exon 15) as determined by qPCR. Results are normalized on GAPDH levels and expressed as fold difference between DOX+ and DOX− conditions with corresponding P values (Student’s t -test). The bar plots display the mean values of experimental triplicates, with error bars representing the standard deviation. E Agarose gel electrophoresis images of PCR products spanning from exon 14 to 16 and allowing for exon 15 inclusion show a higher abundance of alternative BRD9 transcript in SF3B1 K700E DOX+. For a complete gel image see Supplementary Fig. . F Assessment of BRD9 splice variants in SF3B1 WT and SF3B1 MUT CLL, AML, and UVM cell lines. MEC1, PGA1, and HG3 are SF3B1 WT CLL cell lines, CII an SF3B1 K666E CLL cell line, HNT34 an SF3B1 K700E AML cell line, and MEL202 an SF3B1 R625G UVM cell line. The bar plots display the expression levels of BRD9 transcripts in general, the regular BRD9 transcript, and the alternative BRD9 transcript as determined by qPCR. Results for BRD9 transcripts in general are normalized on GAPDH levels, whereas regular and alternative BRD9 transcript levels are normalized on levels of BRD9 transcripts in general and expressed as fold difference between MEC1 and other cell lines with corresponding P values (Student’s t -test). The bar plots display the mean values of experimental triplicates, with error bars representing the standard deviation. G Agarose gel electrophoresis image of PCR products spanning from exon 14 to 16 and allowing for exon 15 inclusion shows a higher abundance of alternative BRD9 transcript in SF3B1 MUT cell lines. For a complete gel image see Supplementary Fig. . H Sashimi plots from the SF3B1 WT CLL cell line HG3 and the SF3B1 MUT AML and UVM cell lines, HNT34 and MEL202, respectively, illustrating the alternatively spliced exon 15 in BRD9 . The plots for HG3 are based on direct long-read RNA-seq, and HNT34 (SRR8616208) and MEL202 (SRR12354765) on short-read RNA-seq. I BRD9 splice isoform expression in SF3B1 WT and SF3B1 MUT CLL cell lines determined by isoform-specific antibodies. An antibody specific to the C-terminus of the regular BRD9 isoform detected a single band in both SF3B1 WT and SF3B1 MUT cell lines. Conversely, an antibody targeting a constant epitope within the central part of BRD9, detected two bands in the SF3B1 MUT cell line CII and one band in the SF3B1 WT cell lines MEC1 and PGA1, corresponding to the regular higher molecular weight BRD9 isoform and the alternative lower molecular weight BRD9 isoform. For complete blot images see Supplementary Fig. . WT: wildtype; MUT: mutated; DOX: doxycycline.

Techniques: Construct, Over Expression, Control, Expressing, Standard Deviation, Alternative Splicing, Agarose Gel Electrophoresis, RNA Sequencing, Molecular Weight

A Scheme illustrating BRD9 splice variants and predicted protein isoforms according to NCBI RefSeq (Annotation release GCF_000001405.40-RS_2023_03). The zoom-in view highlights the predicted protein sequence stemming from exon 15 inclusion in BRD9 . Upon translation of exon 15, a stop codon emerges near the end of the exon, resulting in a shorter splice isoform with an alternative C-terminus. B Scheme depicting lentiviral constructs for overexpression of the regular and alternative BRD9 splice variants. The corresponding DNA and protein sequences are available in Supplementary Information Appendix 1. C Heatmap illustrating the detected ncBAF complex subunits upon co-immunoprecipitation and subsequent mass spectrometry analysis with the regular or alternative BRD9 isoforms stably overexpressed in the HEK293T cell line. The relative protein levels are based on the total number of identified peptide-spectrum matches (PSMs) for the corresponding protein from the mass spectrometry analysis. The ncBAF complex model to the right of the heatmap illustrates that the alternative BRD9 isoform precipitated a majority of the ncBAF complex subunits. ACTB was not detected among the co-immunoprecipitates. The control cells expressing FLAG-V5-tagged YFP were used. D Volcano plot displaying differential interaction analysis of proteins that selectively interacted more with the regular or alternative BRD9 isoforms (multiple Student’s t -tests with Benjamini–Hochberg multiple testing correction). The top 5 candidates are highlighted in red. E Venn diagram showing a comparison of the significant differentially interacting proteins with the regular or alternative BRD9 isoforms and BRD9-interacting proteins reported by Gaudio et al. . The intersecting proteins are SPEN, BRCA2, and CHD9. F Western blot analysis of V5-tag and BICRA co-immunoprecipitates in the HEK293T cell line with stable overexpression of the regular and alternative BRD9 isoforms. In the V5-tag-immunoprecipitations, both overexpressed BRD9 isoforms precipitated BICRA together with SMARCA4 and SMARCC1. Reciprocal BICRA co-immunoprecipitations precipitated both overexpressed BRD9 isoforms, SMARCA4 and SMARCC1. The control cells were transduced with an empty lentiviral vector. For complete blot images see Supplementary Fig. . G Bar plots showing V5-tag and BICRA co-immunoprecipitation efficiency with BRD9, BICRA, SMARCA4, and SMARCC1. Results are expressed as fold differences between the regular and alternative BRD9 isoforms with corresponding P values (Student’s t -test). The bar plots display the mean values from three repeated experiments, with error bars representing the standard deviation. For detailed quantification and calculation see Supplementary Fig. . PSM: peptide-spectrum matches; FDR: false discovery rate; IP: immunoprecipitation.

Journal: Leukemia

Article Title: The non-canonical BAF chromatin remodeling complex is a novel target of spliceosome dysregulation in SF3B1 -mutated chronic lymphocytic leukemia

doi: 10.1038/s41375-024-02379-4

Figure Lengend Snippet: A Scheme illustrating BRD9 splice variants and predicted protein isoforms according to NCBI RefSeq (Annotation release GCF_000001405.40-RS_2023_03). The zoom-in view highlights the predicted protein sequence stemming from exon 15 inclusion in BRD9 . Upon translation of exon 15, a stop codon emerges near the end of the exon, resulting in a shorter splice isoform with an alternative C-terminus. B Scheme depicting lentiviral constructs for overexpression of the regular and alternative BRD9 splice variants. The corresponding DNA and protein sequences are available in Supplementary Information Appendix 1. C Heatmap illustrating the detected ncBAF complex subunits upon co-immunoprecipitation and subsequent mass spectrometry analysis with the regular or alternative BRD9 isoforms stably overexpressed in the HEK293T cell line. The relative protein levels are based on the total number of identified peptide-spectrum matches (PSMs) for the corresponding protein from the mass spectrometry analysis. The ncBAF complex model to the right of the heatmap illustrates that the alternative BRD9 isoform precipitated a majority of the ncBAF complex subunits. ACTB was not detected among the co-immunoprecipitates. The control cells expressing FLAG-V5-tagged YFP were used. D Volcano plot displaying differential interaction analysis of proteins that selectively interacted more with the regular or alternative BRD9 isoforms (multiple Student’s t -tests with Benjamini–Hochberg multiple testing correction). The top 5 candidates are highlighted in red. E Venn diagram showing a comparison of the significant differentially interacting proteins with the regular or alternative BRD9 isoforms and BRD9-interacting proteins reported by Gaudio et al. . The intersecting proteins are SPEN, BRCA2, and CHD9. F Western blot analysis of V5-tag and BICRA co-immunoprecipitates in the HEK293T cell line with stable overexpression of the regular and alternative BRD9 isoforms. In the V5-tag-immunoprecipitations, both overexpressed BRD9 isoforms precipitated BICRA together with SMARCA4 and SMARCC1. Reciprocal BICRA co-immunoprecipitations precipitated both overexpressed BRD9 isoforms, SMARCA4 and SMARCC1. The control cells were transduced with an empty lentiviral vector. For complete blot images see Supplementary Fig. . G Bar plots showing V5-tag and BICRA co-immunoprecipitation efficiency with BRD9, BICRA, SMARCA4, and SMARCC1. Results are expressed as fold differences between the regular and alternative BRD9 isoforms with corresponding P values (Student’s t -test). The bar plots display the mean values from three repeated experiments, with error bars representing the standard deviation. For detailed quantification and calculation see Supplementary Fig. . PSM: peptide-spectrum matches; FDR: false discovery rate; IP: immunoprecipitation.

Techniques: Sequencing, Construct, Over Expression, Immunoprecipitation, Mass Spectrometry, Stable Transfection, Control, Expressing, Comparison, Western Blot, Transduction, Plasmid Preparation, Standard Deviation

A Volcano plot depicting differentially expressed genes between 18 SF3B1 MUT and 17 SF3B1 WT subset #2 cases with 22 downregulated and 55 upregulated genes ( | log 2 FC | ≥ 0.58 and FDR < 0.01). B Unsupervised clustermap illustrating the distinct clustering of SF3B1 MUT and SF3B1 WT subset #2 cases based on differential gene expression. The four neighboring genes, NOL9 , TAS1R1 , ZBTB48 , and KLHL21 , located on chromosome 1 are depicted in color. The Ward method and the Euclidean metric were employed. C Karyoplot of chromosome 1 displaying differentially expressed genes between SF3B1 MUT and SF3B1 WT subset #2 cases. Examination of chromosomal positions revealed four neighboring genes, NOL9 , TAS1R1 , ZBTB48 , and KLHL21 . D Venn diagram showing the overlap of genes with differential expression between SF3B1 MUT and SF3B1 WT cases in three independent datasets; subset #2 CLL, ICGC CLLE-ES [ , ], and TCGA CLL [ , , ]. For the differential gene expression analyses of the latter two datasets see Supplementary Fig. . ZBTB48 appeared upregulated in all three datasets, while TAS1R1 in subset #2 CLL and ICGC CLLE-ES. E Venn diagram showing the overlap of genes with higher expression levels in SF3B1 MUT subset #2 cases and increased chromatin accessibility in SF3B1 MUT CLL cases from Beekman et al. . The overlap comprises NOL9 , TAS1R1 , ZBTB48 , and KLHL21 . F Coverage tracks from ChIP-seq data sourced from the ENCODE database [ , ], based on the CML cell line K562, illustrating the binding of BRD9 and the ncBAF complex subunits SMARCA4 and SMARCC2 to the gene quartet region on chromosome 1. The promoters for these genes appear active based on the signal for H3K27ac, H3K4me3, H3K9ac, and H3K79me2, and transcriptionally active based on the H3K36me3 signal, whereas transcriptional repression is low based on the low signal for H3K27me3 and H3K9me3. G Gene essentiality map showing the relationship between efficacy and selectivity of genes in shinyDepMap [ , ]. SF3B1 appears as a non-selective dependency with high efficacy, while BRD9 as a selective dependency with high efficacy. BRD9 is in the dependency region of PIK3R1 , EZH2 , and FBXW7 . H Functional similarity clustering showing a chromatin remodeling dependency cluster connected to the BRD9 dependency signature in shinyDepMap. BRD9 clusters with SMARCD1 , a BAF complex subunit, subunits of the Mediator complex, p300/CBP subunits, and subunits of the SAGA complex. I BRD9 dependency ranking of the 1086 DepMap (22Q2) cell lines. For CLL cell lines, gene effect scores are given within the parenthesis. WT: wildtype; MUT: mutated; FDR: false discovery rate; FC: fold change.

Journal: Leukemia

Article Title: The non-canonical BAF chromatin remodeling complex is a novel target of spliceosome dysregulation in SF3B1 -mutated chronic lymphocytic leukemia

doi: 10.1038/s41375-024-02379-4

Figure Lengend Snippet: A Volcano plot depicting differentially expressed genes between 18 SF3B1 MUT and 17 SF3B1 WT subset #2 cases with 22 downregulated and 55 upregulated genes ( | log 2 FC | ≥ 0.58 and FDR < 0.01). B Unsupervised clustermap illustrating the distinct clustering of SF3B1 MUT and SF3B1 WT subset #2 cases based on differential gene expression. The four neighboring genes, NOL9 , TAS1R1 , ZBTB48 , and KLHL21 , located on chromosome 1 are depicted in color. The Ward method and the Euclidean metric were employed. C Karyoplot of chromosome 1 displaying differentially expressed genes between SF3B1 MUT and SF3B1 WT subset #2 cases. Examination of chromosomal positions revealed four neighboring genes, NOL9 , TAS1R1 , ZBTB48 , and KLHL21 . D Venn diagram showing the overlap of genes with differential expression between SF3B1 MUT and SF3B1 WT cases in three independent datasets; subset #2 CLL, ICGC CLLE-ES [ , ], and TCGA CLL [ , , ]. For the differential gene expression analyses of the latter two datasets see Supplementary Fig. . ZBTB48 appeared upregulated in all three datasets, while TAS1R1 in subset #2 CLL and ICGC CLLE-ES. E Venn diagram showing the overlap of genes with higher expression levels in SF3B1 MUT subset #2 cases and increased chromatin accessibility in SF3B1 MUT CLL cases from Beekman et al. . The overlap comprises NOL9 , TAS1R1 , ZBTB48 , and KLHL21 . F Coverage tracks from ChIP-seq data sourced from the ENCODE database [ , ], based on the CML cell line K562, illustrating the binding of BRD9 and the ncBAF complex subunits SMARCA4 and SMARCC2 to the gene quartet region on chromosome 1. The promoters for these genes appear active based on the signal for H3K27ac, H3K4me3, H3K9ac, and H3K79me2, and transcriptionally active based on the H3K36me3 signal, whereas transcriptional repression is low based on the low signal for H3K27me3 and H3K9me3. G Gene essentiality map showing the relationship between efficacy and selectivity of genes in shinyDepMap [ , ]. SF3B1 appears as a non-selective dependency with high efficacy, while BRD9 as a selective dependency with high efficacy. BRD9 is in the dependency region of PIK3R1 , EZH2 , and FBXW7 . H Functional similarity clustering showing a chromatin remodeling dependency cluster connected to the BRD9 dependency signature in shinyDepMap. BRD9 clusters with SMARCD1 , a BAF complex subunit, subunits of the Mediator complex, p300/CBP subunits, and subunits of the SAGA complex. I BRD9 dependency ranking of the 1086 DepMap (22Q2) cell lines. For CLL cell lines, gene effect scores are given within the parenthesis. WT: wildtype; MUT: mutated; FDR: false discovery rate; FC: fold change.

Techniques: Gene Expression, Quantitative Proteomics, Expressing, ChIP-sequencing, Binding Assay, Functional Assay

Journal: Leukemia

Article Title: The non-canonical BAF chromatin remodeling complex is a novel target of spliceosome dysregulation in SF3B1 -mutated chronic lymphocytic leukemia

doi: 10.1038/s41375-024-02379-4

Figure Lengend Snippet: A Dose-response analysis of I-BRD9, PROTAC BRD9 Degrader-1, and dBRD9 treatments in 3 SF3B1 WT cell lines, MEC1, PGA1, and HG3 (all CLL), and 3 SF3B1 MUT cell lines, CII (CLL), HNT34 (AML), and MEL202 (UVM). The cell lines were treated with drug concentrations ranging from 0.001 to 50 µM for 3 days, and cell viability was determined by CellTiter-Glo 2.0. Complete cell killing was exclusively observed with I-BRD9 treatment, allowing for the determination of corresponding IC 50 values for each cell line. The HNT34 cell line exhibited sensitivity to all three drugs, thereby allowing for the determination of IC 50 values for all conditions. Dose-response curves are shown with 95% confidence intervals, while individual dots display the mean values of triplicates, with error bars representing the standard deviation. B I-BRD9 sensitivity profile in 958 cancer cell lines from GDSC . C Assessment of proliferation in an SF3B1 WT cell line, PGA1, and SF3B1 MUT cell lines, CII and HNT34, upon treatment with 10 µM I-BRD9, 25 µM PROTAC BRD9 Degrader-1, or 25 µM dBRD9. Cell lines were treated for 3 days with a 5-hour exposure to BrdU at the end, and proliferation was quantified by flow cytometry, measured as BrdU+ cells. Vehicle (DMSO) was used as the negative control for drug treatment, while cells not exposed to BrdU served as the negative control to assess the specificity of the anti-BrdU antibody. The upper left quadrants (BrdU+ cells) in the density plots represent the percentages of proliferating cells. D Bar plot displaying differences in the percentages of proliferating cells compared to negative controls with corresponding P values (one-way ANOVA). The bar plot displays the mean values from two repeated experiments, with error bars representing the standard deviation. E Assessment of apoptosis in the same cell lines and the identical samples as in ( C ). Treatment with 5 µM Camptothecin and vehicle (DMSO) were used as the positive and negative controls, respectively. Apoptosis was evaluated by Annexin V/PI staining and subsequent flow cytometry analysis. The lower left (Annexin V-/PI- cells), upper left (Annexin V+/PI- cells), and upper right (Annexin V+/PI+ cells) quadrants in the density plots represent the percentages of viable, early apoptotic, and late apoptotic cells, respectively. F Stacked bar plot displaying differences in the percentages of viable, early apoptotic, and late apoptotic cells compared to negative controls with corresponding P values (one-way ANOVA). The bar plot displays the mean values from two repeated experiments, with error bars representing the standard deviation. G Bar plot showing cell viability differences as determined by CellTiter-Glo 2.0 in the same cell lines under the same experimental conditions as in panels ( C , E ) with corresponding P values (one-way ANOVA). The bar plot displays the mean values from two repeated experiments in triplicates, with error bars representing the standard deviation. WT: wildtype; MUT: mutated; IC 50 : half-maximal inhibitory concentration; RLU: relative luminescence unit.

Techniques: Standard Deviation, Flow Cytometry, Negative Control, Staining, Concentration Assay

A Dose-response analysis of I-BRD9 treatment in 3 SF3B1 WT (SKL48, SKL152, SKL53) and 3 SF3B1 MUT (SKL147, SKL47, SKL157) primary CLL cell samples. The cell lines were treated with drug concentrations ranging from 0.001 to 50 µM for 2 days, and cell viability was determined by CellTiter-Glo 2.0. Dose-response curves are shown with 95% confidence intervals, while individual dots display the mean values of triplicates, with error bars representing the standard deviation. B Stacked density plots showing apoptosis assessment in the same primary CLL cell samples as in ( A ). Treatment with 5 µM Camptothecin and vehicle (DMSO) were used as the positive and negative controls, respectively. Apoptosis was evaluated by Annexin V/PI staining and subsequent flow cytometry analysis. The lower left (Annexin V-/PI- cells), upper left (Annexin V+/PI- cells), and upper right (Annexin V+/PI+ cells) quadrants in the density plots represent the percentages of viable, early apoptotic, and late apoptotic cells, respectively. C Stacked bar plot displaying differences in the percentages of viable, early apoptotic, and late apoptotic cells compared to negative controls with corresponding P values (Student’s t -test). The bar plot displays the mean values, with error bars representing the standard deviation. WT: wildtype; MUT: mutated; IC 50 : half-maximal inhibitory concentration; RLU: relative luminescence unit.

Journal: Leukemia

Article Title: The non-canonical BAF chromatin remodeling complex is a novel target of spliceosome dysregulation in SF3B1 -mutated chronic lymphocytic leukemia

doi: 10.1038/s41375-024-02379-4

Figure Lengend Snippet: A Dose-response analysis of I-BRD9 treatment in 3 SF3B1 WT (SKL48, SKL152, SKL53) and 3 SF3B1 MUT (SKL147, SKL47, SKL157) primary CLL cell samples. The cell lines were treated with drug concentrations ranging from 0.001 to 50 µM for 2 days, and cell viability was determined by CellTiter-Glo 2.0. Dose-response curves are shown with 95% confidence intervals, while individual dots display the mean values of triplicates, with error bars representing the standard deviation. B Stacked density plots showing apoptosis assessment in the same primary CLL cell samples as in ( A ). Treatment with 5 µM Camptothecin and vehicle (DMSO) were used as the positive and negative controls, respectively. Apoptosis was evaluated by Annexin V/PI staining and subsequent flow cytometry analysis. The lower left (Annexin V-/PI- cells), upper left (Annexin V+/PI- cells), and upper right (Annexin V+/PI+ cells) quadrants in the density plots represent the percentages of viable, early apoptotic, and late apoptotic cells, respectively. C Stacked bar plot displaying differences in the percentages of viable, early apoptotic, and late apoptotic cells compared to negative controls with corresponding P values (Student’s t -test). The bar plot displays the mean values, with error bars representing the standard deviation. WT: wildtype; MUT: mutated; IC 50 : half-maximal inhibitory concentration; RLU: relative luminescence unit.

Techniques: Standard Deviation, Staining, Flow Cytometry, Concentration Assay

Figure 1. Protein levels of BRD9 in human UF tissues and cells. (A) Immunoblot analysis was performed to determine the levels of BRD9 protein in UFs (n = 17) and myometrium tissues (n = 6). (B) The protein levels of BRD9 in UFs and myometrium in (A) were quantified using NIH Image J software (1.53t version) (NIH, Bethesda, MD, USA) and presented as fold changes (F/M). (C) Immunoblot analysis was performed to determine the levels of BRD9 protein in HuLM and UTSM cells. (D) The BRD9 levels in HuLM and UTSM cells in (C) were quantified using NIH Image J software and presented as fold changes (HuLM/UTSM). β-actin was used as an endogenous control. P: patients, M: myometrium; F: uterine fibroids, **** p < 0.0001.

Journal: International journal of molecular sciences

Article Title: Bromodomain-Containing Protein 9 Regulates Signaling Pathways and Reprograms the Epigenome in Immortalized Human Uterine Fibroid Cells.

doi: 10.3390/ijms25020905

Figure Lengend Snippet: Figure 1. Protein levels of BRD9 in human UF tissues and cells. (A) Immunoblot analysis was performed to determine the levels of BRD9 protein in UFs (n = 17) and myometrium tissues (n = 6). (B) The protein levels of BRD9 in UFs and myometrium in (A) were quantified using NIH Image J software (1.53t version) (NIH, Bethesda, MD, USA) and presented as fold changes (F/M). (C) Immunoblot analysis was performed to determine the levels of BRD9 protein in HuLM and UTSM cells. (D) The BRD9 levels in HuLM and UTSM cells in (C) were quantified using NIH Image J software and presented as fold changes (HuLM/UTSM). β-actin was used as an endogenous control. P: patients, M: myometrium; F: uterine fibroids, **** p < 0.0001.

Article Snippet: In addition, the BRD9 inhibitor, I-BRD9, was purchased from Selleck Chemical (Cat# S7835, Houston, TX, USA).

Techniques: Western Blot, Software, Control

Figure 2. Treatments with I-BRD9 decrease UF cell proliferation and ECM levels. (A) HuLM and UTSM cell proliferation was performed in the presence or absence of I-BRD9 with a trypan blue exclusion assay. (B) The protein levels of PCNA and fibronectin (FN) were examined via Immunoblot analysis using anti-PCNA and anti-FN antibodies, respectively. Quantification of immunoblot signals was performed after normalization to β-actin. (C) Flow cytometry analysis was performed to measure the cell cycle phase distribution (blue color: G1; yellow color: S; green color: G2) in HuLM cells treated with I-BRD9 (n = 3 for each group). (D) Quantitative analysis of cell cycle data. (E) Morphological changes to HuLM cells after treatment with I-BRD9. Magnification was applied ×10. Pictures were taken by EVOS XL Core imaging system (Invitrogen) * p < 0.05, ** p < 0.01; *** p < 0.001; **** p < 0.0001. * Comparison between DMSO- and I-BRD9 treated HuLM cells, * comparison between DMSO- and I-BRD9 treated UTSM cells.

Journal: International journal of molecular sciences

Article Title: Bromodomain-Containing Protein 9 Regulates Signaling Pathways and Reprograms the Epigenome in Immortalized Human Uterine Fibroid Cells.

doi: 10.3390/ijms25020905

Figure Lengend Snippet: Figure 2. Treatments with I-BRD9 decrease UF cell proliferation and ECM levels. (A) HuLM and UTSM cell proliferation was performed in the presence or absence of I-BRD9 with a trypan blue exclusion assay. (B) The protein levels of PCNA and fibronectin (FN) were examined via Immunoblot analysis using anti-PCNA and anti-FN antibodies, respectively. Quantification of immunoblot signals was performed after normalization to β-actin. (C) Flow cytometry analysis was performed to measure the cell cycle phase distribution (blue color: G1; yellow color: S; green color: G2) in HuLM cells treated with I-BRD9 (n = 3 for each group). (D) Quantitative analysis of cell cycle data. (E) Morphological changes to HuLM cells after treatment with I-BRD9. Magnification was applied ×10. Pictures were taken by EVOS XL Core imaging system (Invitrogen) * p < 0.05, ** p < 0.01; *** p < 0.001; **** p < 0.0001. * Comparison between DMSO- and I-BRD9 treated HuLM cells, * comparison between DMSO- and I-BRD9 treated UTSM cells.

Article Snippet: In addition, the BRD9 inhibitor, I-BRD9, was purchased from Selleck Chemical (Cat# S7835, Houston, TX, USA).

Techniques: Trypan Blue Exclusion Assay, Western Blot, Flow Cytometry, Imaging, Comparison

Figure 3. Treatment with I-BRD9 sculpts the transcriptome of UF cells. (A) Venn diagrams demon- strating the overlap of DEGs identified via three methods of Limma + voom, edgeR, and DESeq2

Journal: International journal of molecular sciences

Article Title: Bromodomain-Containing Protein 9 Regulates Signaling Pathways and Reprograms the Epigenome in Immortalized Human Uterine Fibroid Cells.

doi: 10.3390/ijms25020905

Figure Lengend Snippet: Figure 3. Treatment with I-BRD9 sculpts the transcriptome of UF cells. (A) Venn diagrams demon- strating the overlap of DEGs identified via three methods of Limma + voom, edgeR, and DESeq2

Article Snippet: In addition, the BRD9 inhibitor, I-BRD9, was purchased from Selleck Chemical (Cat# S7835, Houston, TX, USA).

Techniques:

Figure 4. I-BRD9 altered cell cycle- and apoptosis-related gene expression in HuLM cells. RNA-seq revealed the downregulation of CCND1, CCND3, CDK2, CDK6, PCNA, and BCL-2, and upregulation of CDKN1C and CDKN1B. * p < 0.05; **** p < 0.0001.

Journal: International journal of molecular sciences

Article Title: Bromodomain-Containing Protein 9 Regulates Signaling Pathways and Reprograms the Epigenome in Immortalized Human Uterine Fibroid Cells.

doi: 10.3390/ijms25020905

Figure Lengend Snippet: Figure 4. I-BRD9 altered cell cycle- and apoptosis-related gene expression in HuLM cells. RNA-seq revealed the downregulation of CCND1, CCND3, CDK2, CDK6, PCNA, and BCL-2, and upregulation of CDKN1C and CDKN1B. * p < 0.05; **** p < 0.0001.

Article Snippet: In addition, the BRD9 inhibitor, I-BRD9, was purchased from Selleck Chemical (Cat# S7835, Houston, TX, USA).

Techniques: Gene Expression, RNA Sequencing

Figure 5. I-BRD9 altered the RNA expression of ECM-related genes in HuLM cells. RNA-seq revealed the upregulation of MMP2, MMP11, MMP15, MMP16, and MMP17 and downregulation of COL13A1, COL16A1, and Col17A1 in HuLM cells treated with I-BRD9. *** p < 0.001; **** p < 0.0001.

Journal: International journal of molecular sciences

Article Title: Bromodomain-Containing Protein 9 Regulates Signaling Pathways and Reprograms the Epigenome in Immortalized Human Uterine Fibroid Cells.

doi: 10.3390/ijms25020905

Figure Lengend Snippet: Figure 5. I-BRD9 altered the RNA expression of ECM-related genes in HuLM cells. RNA-seq revealed the upregulation of MMP2, MMP11, MMP15, MMP16, and MMP17 and downregulation of COL13A1, COL16A1, and Col17A1 in HuLM cells treated with I-BRD9. *** p < 0.001; **** p < 0.0001.

Article Snippet: In addition, the BRD9 inhibitor, I-BRD9, was purchased from Selleck Chemical (Cat# S7835, Houston, TX, USA).

Techniques: RNA Expression, RNA Sequencing

Figure 6. Enrichment analysis for histone modifications. The dot plots showed the top twenty enrichment terms for histone modification associated with up DEGs (A) and down DEGs (B) in response to I-BRD9 treatment. The x-axis represents the gene ratio, and the y-axis describes the enrichment components. The area of the circle is proportional to the number of genes assigned to the term, and the color accords with the adjusted p-value. (C) The levels of histone marks, including H3K4me3, H3K27me3, H3K9Ac, and H3K18Ac, were examined via immunoblot analysis in HuLM cells in the presence (I-BRD9 (1–5 µM) or absence (DMSO) of I-BRD9. (D) The levels of H3K4me3 were quantified in HuLM cells in the presence or absence of I-BRD9 using NIH Image J.

Journal: International journal of molecular sciences

Article Title: Bromodomain-Containing Protein 9 Regulates Signaling Pathways and Reprograms the Epigenome in Immortalized Human Uterine Fibroid Cells.

doi: 10.3390/ijms25020905

Figure Lengend Snippet: Figure 6. Enrichment analysis for histone modifications. The dot plots showed the top twenty enrichment terms for histone modification associated with up DEGs (A) and down DEGs (B) in response to I-BRD9 treatment. The x-axis represents the gene ratio, and the y-axis describes the enrichment components. The area of the circle is proportional to the number of genes assigned to the term, and the color accords with the adjusted p-value. (C) The levels of histone marks, including H3K4me3, H3K27me3, H3K9Ac, and H3K18Ac, were examined via immunoblot analysis in HuLM cells in the presence (I-BRD9 (1–5 µM) or absence (DMSO) of I-BRD9. (D) The levels of H3K4me3 were quantified in HuLM cells in the presence or absence of I-BRD9 using NIH Image J.

Article Snippet: In addition, the BRD9 inhibitor, I-BRD9, was purchased from Selleck Chemical (Cat# S7835, Houston, TX, USA).

Techniques: Modification, Western Blot

Figure 7. I-BRD9 altered the RNA expression of epigenetic regulators in HuLM cells. RNA-seq revealed the downregulation of EZH2, SUV39H1, SUV39H2, DNMT3B, and DNMT1 and upregulation of SIRT2 in HuLM cells treated with I-BRD9. *** p < 0.001; **** p < 0.0001.

Journal: International journal of molecular sciences

Article Title: Bromodomain-Containing Protein 9 Regulates Signaling Pathways and Reprograms the Epigenome in Immortalized Human Uterine Fibroid Cells.

doi: 10.3390/ijms25020905

Figure Lengend Snippet: Figure 7. I-BRD9 altered the RNA expression of epigenetic regulators in HuLM cells. RNA-seq revealed the downregulation of EZH2, SUV39H1, SUV39H2, DNMT3B, and DNMT1 and upregulation of SIRT2 in HuLM cells treated with I-BRD9. *** p < 0.001; **** p < 0.0001.

Article Snippet: In addition, the BRD9 inhibitor, I-BRD9, was purchased from Selleck Chemical (Cat# S7835, Houston, TX, USA).

Techniques: RNA Expression, RNA Sequencing

Figure 8. The expression of m6A writers in HuLM cells in response to I-BRD9 treatments and the experimental model. The RNA expression of METTL3 (A) in HuLM cells treated with I-BRD9. (B) The comparison of protein levels of m6A readers (YTHDC1 and YTHDF2) in vehicle- and I- BRD9-treated HuLM cells. (C) The experimental model shows that I-BRD9 treatment promotes apoptosis, induces cell cycle arrest, represses ECM accumulation, and reprograms the epigenome and epitranscriptome in UF cells. Figure 8C was created using BioRender software (BioRender.com, accessed on 23 December 2023). **** p < 0.0001.

Journal: International journal of molecular sciences

Article Title: Bromodomain-Containing Protein 9 Regulates Signaling Pathways and Reprograms the Epigenome in Immortalized Human Uterine Fibroid Cells.

doi: 10.3390/ijms25020905

Figure Lengend Snippet: Figure 8. The expression of m6A writers in HuLM cells in response to I-BRD9 treatments and the experimental model. The RNA expression of METTL3 (A) in HuLM cells treated with I-BRD9. (B) The comparison of protein levels of m6A readers (YTHDC1 and YTHDF2) in vehicle- and I- BRD9-treated HuLM cells. (C) The experimental model shows that I-BRD9 treatment promotes apoptosis, induces cell cycle arrest, represses ECM accumulation, and reprograms the epigenome and epitranscriptome in UF cells. Figure 8C was created using BioRender software (BioRender.com, accessed on 23 December 2023). **** p < 0.0001.

Article Snippet: In addition, the BRD9 inhibitor, I-BRD9, was purchased from Selleck Chemical (Cat# S7835, Houston, TX, USA).

Techniques: Expressing, RNA Expression, Comparison, Software

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