FIG 2.
KLF4 is dispensable for brown adipogenesis in culture. (A to C) Klf4f/f immortalized brown preadipocytes were infected with retroviral vector MSCVpuro expressing Cre. (A) Western blot analysis of KLF4 before differentiation. The asterisk indicates a nonspecific band. RbBP5 was used as a loading control. (B) Oil Red O staining at day 7 of adipogenesis. Upper panels, stained dishes; lower panels, representative fields under the microscope. (C) qRT-PCR analysis of Klf4, adipogenesis markers (Pparg, Cebpa, and Fabp4) and brown adipocyte markers (Prdm16 and Ucp1) at indicated time points of adipogenesis. D0, day 0. (D, E) Klf4f/f immortalized brown preadipocytes were infected with adenoviruses expressing GFP (Ad-GFP) or Cre (Ad-Cre). Two days later, cells were replated, followed by adipogenesis assays. (D) Oil Red O staining. (E) qRT-PCR analysis of gene expression. (F to H) Klf4f/f primary brown preadipocytes were infected with Ad-GFP or Ad-Cre, followed by adipogenesis assays. (F) Oil Red O staining. (G) qRT-PCR analysis of gene expression. (H) D7 mature brown adipocytes were treated with 100 nM CL-316,243 for 4 h, followed by qRT-PCR analysis of Ucp1 expression.