Fig. 2. BAF subunits SMARCA4, SMARCB1 and ARID1A are required for adipogenesis.
a–c Depletion of SMARCA4 by auxin-inducible degron (AID) system inhibits adipogenesis. SV40T-immortalized Smarca4AID/AID brown preadipocytes were infected with retroviruses expressing Tir1 or vector (Vec), and then treated with auxin for indicated times. a A schematic illustration of the auxin-induced depletion of endogenous SMARCA4 protein in Tir1-expressing Smarca4AID/AID cells. TIR1, transport inhibitor response 1; Cul1, Cullin-1; Rbx1, RING-box protein 1; Skp1, S-phase kinase-associated protein 1. b Western blot analyses using antibodies indicated on the left. RbBP5 was used as a loading control. n = 3 biological replicates. c Oil Red O staining at day 7 (D7) of adipogenesis. Cells were treated with auxin throughout the differentiation. n = 2 biological replicates. Scale bar = 50 μm. d SMARCB1 is essential for brown adipogenesis in vivo. Sections of the interscapular area of E18.5 Smarcb1f/f (f/f) and Smarcb1f/f;PdgfRα-Cre (cKO) embryos were stained with H&E (left panels) or with antibodies recognizing brown adipose tissue (BAT) marker Ucp1 (green) and skeletal muscle marker Myosin (red) (right panels). B, BAT; M, muscle. Scale bar = 1 mm. n = 2 biological replicates. e–h SMARCB1 is essential for adipogenesis in culture. SV40T-immortalized Smarcb1f/f brown preadipocytes were infected with adenoviral GFP or Cre, followed by adipogenesis assays. e Deletion of Smarcb1 does not affect cell growth rates of immortalized brown preadipocytes. 1 ×105 preadipocytes were plated and the cumulative cell numbers were determined for 5 days (n = 2). Data are presented as mean values ± std. dev. f Before (D-3) and during (D2) adipogenesis, nuclear extracts were analyzed by Western blot using indicated antibodies. n = 2 biological replicates. g Oil Red O staining at D7 of adipogenesis. n = 3 biological replicates. Scale bar = 50 μm. h Expression of Cebpb, Pparg, Cebpa and Fabp4 before (D-3) and during (D2) adipogenesis was determined using RNA-Seq (n = 1). RPKM values indicate gene expression levels. i–k ARID1A is required for adipogenesis in culture. Immortalized Smarcb1f/f brown preadipocytes were infected with lentiviral vectors expressing control (shCtrl) or Arid1a knockdown shRNA (shArid1a), followed by adipogenesis assays. i Western blot analysis of ARID1A in preadipocytes. n = 2 biological replicates. j Oil Red O staining at D7 of adipogenesis. n = 3 biological replicates. Scale bar = 50 μm. k qRT-PCR analysis of Cebpb, Pparg, and Fabp4 expression at indicated time points of adipogenesis. Expression levels were normalized to 18 S rRNA. n = 2 biologically independent samples. Data are presented as mean values ± std. dev.