Journal: Journal of Lipid Research

Article Title: Chromatin remodeler BRD9 represses transcription of PPARα target genes, including CPT1A to suppress lipid metabolism

doi: 10.1016/j.jlr.2025.100874

Figure Lengend Snippet: Inhibition of BRD9 enhances the induction of PPARα-downstream gene CPT1A by WY-14643. A: Schematic image of mammalian cBAF, PBAF, and ncBAF . B: Glycerol gradient density sedimentation of the nuclear extract of HepG2 cells. Subunits of each SWI/SNF complex and PPARα protein were detected by Western blot analysis (C–G) HepG2, PHH, PMH, or HepaSH cells were pre-treated with 20 μM BI-9564. After 12 h, the cells were co-treated with 10 μM WY-14643 and 20 μM BI-9564 for 48 h. Human (C and E) and mouse (F) CPT1A mRNA levels were determined using real-time RT-PCR. (D) CPT1A, PPARα, and GAPDH protein levels in HepG2 cells were determined by Western blotting. Each column represents the mean ± SD (n = 4). Western blot experiments were conducted with three independent replicates. In panel (C), data were analyzed using one-way ANOVA followed by the Games-Howell test. In panel (F), data were analyzed using the Kruskal–Wallis test followed by Dunn’s test. Data in all other panels were analyzed by one-way ANOVA followed by Tukey’s test. ∗ 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 BI-9564 (−). NT: non-treatment.

Article Snippet: Rabbit polyclonal antibody for human BRD9 (A303-781A) was purchased from Bethyl Laboratories.

Techniques: Inhibition, Sedimentation, Western Blot, Quantitative RT-PCR

Journal: Journal of Lipid Research

Article Title: Chromatin remodeler BRD9 represses transcription of PPARα target genes, including CPT1A to suppress lipid metabolism

doi: 10.1016/j.jlr.2025.100874

Figure Lengend Snippet: Effects of BRD7 and BRD9 knockdown on CPT1A expression in WY-14643-treated HepG2 cells. A–C: HepG2 cells were transfected with siRNA against BRD7 (siBRD7) or BRD9 (siBRD9). After 24 h, the cells were treated with 30 μM WY-14643 for 48 h. A: BRD7, BRD9, PPARα, and GAPDH protein levels were determined using Western blot analysis. B and C: CPT1A mRNA and protein levels were determined using real-time RT-PCR and Western blotting analysis, respectively. Each column represents the mean ± SD (n = 4). Western blot experiments were conducted with three independent replicates. Data in all panels were analyzed by one-way ANOVA followed by Tukey’s test. ∗∗ P < 0.01 and ∗∗∗ P < 0.001, compared with NT, † P < 0.05 and ††† P < 0.001, compared with siControl. NT: non-treatment.

Article Snippet: Rabbit polyclonal antibody for human BRD9 (A303-781A) was purchased from Bethyl Laboratories.

Techniques: Knockdown, Expressing, Transfection, Western Blot, Quantitative RT-PCR

Journal: Journal of Lipid Research

Article Title: Chromatin remodeler BRD9 represses transcription of PPARα target genes, including CPT1A to suppress lipid metabolism

doi: 10.1016/j.jlr.2025.100874

Figure Lengend Snippet: BRD9 directly interacts with PPARα. A: Co-immunoprecipitation assay to examine the interaction between PPARα and BRD9 was performed. HEK293T cells were transfected with FLAG-PPARα plasmid together with the BRD7-His or BRD9-His plasmids using Lipofectamine 3000. Cell lysates were subjected to co-immunoprecipitation with His-tag antibody. B: Direct interaction between PPARα and BRD9. HEK293T cells were individually transfected with FLAG-PPARα, BRD7-His, or BRD9-His plasmids. Affinity purification was conducted using cell lysates, and the purified FLAG-PPAR protein was incubated with purified BRD7/9-His proteins. C: Western blotting to analyze lysine acetylation of PPARα. Lysate of FLAG-PPARα plasmid-transfected HEK293T cells was subjected to Western blotting using anti-acetylated lysine and anti-FLAG antibodies. A smaller amount of protein (10 μg) was loaded compared to A (30 μg). Western blot experiments were conducted with three independent replicates.

Article Snippet: Rabbit polyclonal antibody for human BRD9 (A303-781A) was purchased from Bethyl Laboratories.

Techniques: Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Affinity Purification, Purification, Incubation, Western Blot

Journal: Journal of Lipid Research

Article Title: Chromatin remodeler BRD9 represses transcription of PPARα target genes, including CPT1A to suppress lipid metabolism

doi: 10.1016/j.jlr.2025.100874

Figure Lengend Snippet: Effects of WY-14643 and/or BI-9564 treatment on the binding of PPARα and BRD9 and on the chromatin accessibility around the CPT1A intronic peroxisomal proliferator response element (PPRE). A: Scheme of the CPT1A gene. B: Binding of PPARα to CPT1A intronic PPRE. HepG2 cells were pre-treated with 20 μM BI-9564. After 12 h, the cells were co-treated with 10 μM WY-14643 and 20 μM BI-9564 for 48 h. A ChIP assay using an anti-PPARα antibody was performed, and the purified DNA was analyzed by real-time PCR targeting CPT1A intronic PPRE. C: The interaction between PPARα and BRD9. HEK293T cells were transfected with FLAG-PPARα and BRD9-His plasmids using Lipofectamine 3000, and treated with 30 μM WY-14643 and/or 40 μM BI-9564 for 48 h. The cell lysates were co-immunoprecipitated with anti-His-tag antibody. FLAG-PPARα and BRD9-His were detected by Western blotting. D: Chromatin accessibility around CPT1A intronic PPRE. HepG2 cells were pre-treated with 20 μM BI-9564. After 12 h, the cells were co-treated with 10 μM WY-14643 and 20 μM BI-9564 for 48 h; a FAIRE assay was then performed on the cells. Purified DNA was analyzed by real-time PCR targeting the CPT1A intronic PPRE. Each column represents the mean ± SD (n = 3). Data in all panels were analyzed by one-way ANOVA followed by Tukey’s test. ∗∗∗ P < 0.001, compared with NT; † P < 0.05 and ††† P < 0.001, compared with BI-9564 (−). NT: non-treatment.

Article Snippet: Rabbit polyclonal antibody for human BRD9 (A303-781A) was purchased from Bethyl Laboratories.

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