ibrd9  (Cayman Chemical)

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: Nature communications

Article Title: BRD9-mediated chromatin remodeling suppresses osteoclastogenesis through negative feedback mechanism.

doi: 10.1038/s41467-023-37116-5

Figure Lengend Snippet: Fig. 3 | BRD9 deletion and inhibition in BMDMs leads to overactivated osteo- clastogenesis in vitro. a TRAP staining and quantification analysis of BMDMs from 4-week-old LysM-Cre;Brd9fl/flmice and littermate control mice after 3 days of RANKL-induction. Scale bar, 200 μm. n = 8. b The mRNA expression of osteoclastic- specific genes of Acp5, Ctsk, and Mmp9 in BMDMs from LysM-Cre;Brd9fl/flmice compared with that from control littermates after 3 days of RANKL-induction, as measured by qPCR. n = 3 for LysM-Cre;Brd9fl/flmice group. n = 6 for control litter- mates. c The protein expression of MMP9 and CTSK in BMDMs from LysM- Cre;Brd9fl/flmice compared with thatfrom control littermates after 3 days of RANKL- induction, as measured by western blot. d Cell viability of RANKL-induced BMDMs with 1 day of iBRD9 at different concentration, shown by cell counting kit 8 assay. n = 10 biologically independent samples. e The mRNA expression of Acp5 and Mmp9 in RANKL-induced BMDMs with 1 day of iBRD9 at different concentration, as measured by qPCR. n = 5 biologically independent samples for Acp5. n = 3

Article Snippet: For the BRD9 inhibitor/degrader experiments, the cells were treated with either vehicle or varying concentrations of iBRD9 (Tocris Bioscience, 5591) or dBRD9 (R&D Systems, 6606) twenty-four hours after plated, as well as IFN-β1 cytokine (0.0625 ng/ml, R&D Systems, 8234-MB), DEX (10−11−10−9M), JQ1 (0.25μM, Abmole Bioscience, M2167) and ZOL (10μM, Abmole Bioscience, M5032).

Techniques: Inhibition, In Vitro, Staining, Control, Expressing, Western Blot, Concentration Assay, Cell Counting

Journal: Nature Communications

Article Title: ARID1A safeguards the canalization of the cell fate decision during osteoclastogenesis

doi: 10.1038/s41467-024-50225-z

Figure Lengend Snippet: a The protein expression of ARID1A and BRD9 in BMCs from 4-week-old male LysM-Cre;Arid1a fl/fl mice and littermate control mice after RANKL-induction, as measured by western blot. b GSEA analysis of DEG profiles in bulk RNA-seq between the control group and LysM-Cre;Arid1a fl/f group. c The mRNA expression of Stat1 and Ifnb1 in BMCs from 4-week-old male LysM-Cre;Arid1a fl/fl mice and littermate control mice after RANKL-induction, as measured by qPCR. n = 3 biologically independent samples. d TRAP staining and quantification analysis of iBRD9 or control vector treated BMCs from 4-week-old male LysM-Cre;Arid1a fl/fl mice and littermate control mice after RANKL-induction. Scale bar, 100 μm. n = 5 biologically independent samples. e The mRNA expression of Mmp9, Acp5 and Dcstamp and in iBRD9 or control vector treated BMCs from 4-week-old male LysM-Cre;Arid1a fl/fl mice and littermate control mice after RANKL-induction. n = 4 biologically independent samples for Mmp9 and Acp5 . n = 3 biologically independent samples in Dcstamp . f The protein expression of ARID1A, MMP9, and CTSK in iBRD9 or control vector treated BMCs from 4-week-old male LysM-Cre;Arid1a fl/fl mice and littermate control mice after RANKL-induction, as measured by western blot. All data in this figure are represented as mean ± SD. Two-tailed Student’s t -test for c . One-way ANOVA with Tukey’s multiple comparisons test for d and e . All experiments were performed in triplicates unless otherwise stated. Source data and exact p values are provided in the Source data file.

Article Snippet: For the inhibitor experiments, the cells were treated with either vehicle or iBRD9 (1 μM, Tocris Bioscience, 5591), JQ1 (0.08 μM, Abmole Bioscience, M2167), and SP-141 (0.06 μM, Csnpharm, 223123) twenty-four hours after plated.

Techniques: Expressing, Control, Western Blot, RNA Sequencing, Staining, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: ARID1A safeguards the canalization of the cell fate decision during osteoclastogenesis

doi: 10.1038/s41467-024-50225-z

Figure Lengend Snippet: a The protein expression of STAT1 and CTSK and b STAT1 immunofluorescence (green) in JQ1 or control vector treated BMCs from 4-week-old male LysM-Cre;Arid1a fl/fl mice and littermate control mice after RANKL-induction. c GSEA analysis of DEG profiles in bulk RNA-seq between JQ1 or control vector treated BMCs after RANKL-induction. d Co-IP assay with BRD9 antibody (or IgG) in BMCs during osteoclastic induction, followed by immunoblotting of BRD4 and BRD9. e The protein expression of ARID1A and STAT1 in JQ1 or control vector treated BMCs from 4-week-old male LysM-Cre;Arid1a fl/fl mice and littermate control mice after RANKL-induction. f The protein expression of ARID1A, MMP9 and NFATc1 in JQ1, iBRD9 or control vector treated BMCs from 4-week-old male LysM-Cre;Arid1a fl/fl mice and littermate control mice after RANKL-induction. All experiments were performed in triplicates unless otherwise stated. Source data are provided in the Source data file.

Article Snippet: For the inhibitor experiments, the cells were treated with either vehicle or iBRD9 (1 μM, Tocris Bioscience, 5591), JQ1 (0.08 μM, Abmole Bioscience, M2167), and SP-141 (0.06 μM, Csnpharm, 223123) twenty-four hours after plated.

Techniques: Expressing, Immunofluorescence, Control, Plasmid Preparation, RNA Sequencing, Co-Immunoprecipitation Assay, Western Blot

Journal: bioRxiv

Article Title: ncBAF, a chromatin remodeler, enhances PXR-mediated transcriptional activation in the human and mouse liver

doi: 10.1101/2023.02.03.527063

Figure Lengend Snippet: Interaction between PXR and BRD9. HEK293T cells were transfected with FLAG-PXR along with BRD7-His or BRD9-His plasmid (A), transfected with BRD9-His along with FLAG-PXR or FLAG-PXR K109A plasmid (B), or transfected with FLAG-PXR and BRD9-His plasmids followed by treatment with rifampicin along with iBRD9 for 1 hr (C). ShP51 cells were treated with rifampicin along with iBRD9 for 24 hr (D). (A, B, and C) Immunoprecipitation with anti-FLAG antibody was performed using whole HEK293T cell lysates, and FLAG and His tags were detected by Western blotting. (D) Immunoprecipitation with anti-PXR antibody was performed using ShP51 cell nuclear extracts, and BRD9 and PXR proteins were detected by Western blotting. IP: immunoprecipitation, WB: Western blotting. The experiments were repeated two times with similar results.

Article Snippet: Simvastatin and iBRD9 were purchased from Toronto Research Chemicals (Toronto, Canada) and Cayman Chemical (Ann Arbor, MI), respectively.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: ncBAF, a chromatin remodeler, enhances PXR-mediated transcriptional activation in the human and mouse liver

doi: 10.1101/2023.02.03.527063

Figure Lengend Snippet: Effects of BRD7 or BRD9 knockdown or BRD9 inhibition on PXR-mediated induction of CYP3A4 expression. (A) ShP51 cells were transfected with siRNA for BRD7 (siBRD7) or BRD9 (siBRD9). After incubation for 24 hr, the cells were treated with 10 μM rifampicin or 10 μM simvastatin. ShP51 cells (B), HepaRG cells (C), and human primary hepatocytes (D and E) were treated with rifampicin or simvastatin along with iBRD9. PXR, RXRα and GAPDH (A and B) protein, CYP3A4 and GAPDH mRNA (A-D), and CYP3A4 enzyme activity (E) were evaluated by Western blotting, real-time RT□PCR, and P450-Glo assay. Each column represents the mean ± SD (n =3-4). n refers to biological repeats. * P < 0.05, ** P < 0.01, and *** P < 0.001, compared with NT, † P < 0.05, †† P < 0.01, and ††† P < 0.001, compared with siControl or iBRD9 (−). NT: non-treatment. The experiments were repeated two times with similar results.

Article Snippet: Simvastatin and iBRD9 were purchased from Toronto Research Chemicals (Toronto, Canada) and Cayman Chemical (Ann Arbor, MI), respectively.

Techniques: Knockdown, Inhibition, Expressing, Transfection, Incubation, Activity Assay, Western Blot, Glo Assay

Journal: bioRxiv

Article Title: ncBAF, a chromatin remodeler, enhances PXR-mediated transcriptional activation in the human and mouse liver

doi: 10.1101/2023.02.03.527063

Figure Lengend Snippet: Effects of iBRD9 on PXR binding to chromatin and changes in chromatin structure. (A) ShP51 cells were treated with 20 μM rifampicin along with 20 μM iBRD9 for 24 hr. SSE analysis followed by Western blotting for BRD9 or PXR was performed. The peaks of band intensity are shown in red. (B) Schematic representation of the upstream of CYP3A4 gene. ShP51 cells were treated with 10 μM rifampicin along with 10 μM iBRD9 for 24 hr. Immunoprecipitation with anti-PXR antibody (C) or anti-BRD9 antibody (D) was performed using chromatin from ShP51 cells. Enrichment of the proximal promoter, distal enhancer, and far enhancer of CYP3A4 was evaluated by real-time PCR. (E) Changes in chromatin structure was evaluated by FAIRE followed by real-time PCR. Each column represents the mean ± SD (n = 3). n refers to biological repeats. * P < 0.05, ** P < 0.01, and *** P < 0.001, compared with NT, †† P < 0.01 and ††† P < 0.001, compared with iBRD9 (−). NT: non-treatment. The experiments were repeated two times with similar results.

Article Snippet: Simvastatin and iBRD9 were purchased from Toronto Research Chemicals (Toronto, Canada) and Cayman Chemical (Ann Arbor, MI), respectively.

Techniques: Binding Assay, Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: ncBAF, a chromatin remodeler, enhances PXR-mediated transcriptional activation in the human and mouse liver

doi: 10.1101/2023.02.03.527063

Figure Lengend Snippet: Effects of iBRD9 on the induction of CYP3A in mouse liver. C57BL/6J or hCYP3A-MAC/hPXR male mice (n = 6) were intraperitoneally treated with 50 mg/kg PCN or 10 mg/kg rifampicin for four consecutive days and intraperitoneally treated with 10 mg/kg iBRD9 every other day. Cyp3a11, Cyp3a25, CYP3A4, and Gapdh mRNA (A, D), and Cyp3a, CYP3A4, and KDEL protein (B, E) levels were evaluated by real-time RT□PCR and Western blotting, respectively. (C, F) Triazolam α- and 4-hydroxylase activities were evaluated as marker activity for Cyp3a and CYP3A4. Each column represents the mean ± SD (n = 6). n refers to biological repeats. ** P < 0.01 and *** P < 0.001, compared with vehicle, † P < 0.05 and †† P < 0.01, compared with iBRD9 (−). The experiments were repeated two times with similar results.

Article Snippet: Simvastatin and iBRD9 were purchased from Toronto Research Chemicals (Toronto, Canada) and Cayman Chemical (Ann Arbor, MI), respectively.

Techniques: Western Blot, Marker, Activity Assay

Journal: bioRxiv

Article Title: ncBAF, a chromatin remodeler, enhances PXR-mediated transcriptional activation in the human and mouse liver

doi: 10.1101/2023.02.03.527063

Figure Lengend Snippet: Effects of iBRD9 treatment on efavirenz-mediated hepatic lipid accumulation and hepatotoxicity in vivo . C57BL/6J male mice fed a low-fat diet for 1 week were orally treated with 20 mg/kg efavirenz and intraperitoneally treated with 20 mg/kg iBRD9 daily for 1 week. (A) Survival rate of mice administered with efavirenz and/or iBRD9. (B) Cyp3a11, Sqle and Cd36 mRNA levels were evaluated by real-time RT□PCR. (C) The ratio of liver weight to body weight. Plasma and hepatic cholesterol and triglyceride (D) and plasma AST and ALT (F) levels were evaluated. (E) Liver specimens were subjected to H&E staining and oil red O staining. Correlation of plasma ALT level with hepatic cholesterol level (G), plasma efavirenz level or plasma 8-hydroxy efavirenz level (K). (H) Cyp2b10 mRNA level was evaluated by real-time RT□PCR. (I) Efavirenz 8-hydroxylase activity was evaluated as a marker activity for Cyp2b10. (J) Plasma 8-hydroxyefavirenz and efavirenz levels were evaluated. Each column represents the mean ± SD (n = 5–6). n refers to biological repeats. * P < 0.05 and *** P < 0.001, compared with vehicle, † P < 0.05 and †† P < 0.01, compared with efavirenz administration. The experiments were repeated two times with similar results.

Article Snippet: Simvastatin and iBRD9 were purchased from Toronto Research Chemicals (Toronto, Canada) and Cayman Chemical (Ann Arbor, MI), respectively.

Techniques: In Vivo, Clinical Proteomics, Staining, Activity Assay, Marker