Fig. 4: Asc-1 regulates white versus beige adipocyte lineage commitment in a subset of proliferating precursors.

a Ppary, Pgc1α, and Prdm16 gene expression of subconfluent proliferating shAsc-1 and shScr preadipocytes (n = 4). b Preadipocytes were grown to 100% confluence and differentiation was induced. RNA was taken from the day of differentiation start (preadipocyte d0) and after 8 days (preadipocyte d8), as well as after 8 days of insulin treatment (insulin), insulin and rosiglitazone treatment (insulin + rosi), or differentiation (adipocyte). Relative gene expression of Ppary (n = 3), Ucp1 (n = 6; for shScr insulin n = 3), Pgc1α (n = 6; for shScr insulin n = 4), and Prdm16 (n = 6; for shScr insulin n = 4). c Western blot of UCP1 (33 kDa), PPARy (54 kDa), and β-actin (42 kDa; n = 1). d shScr preadipoctyes were plated in presence of 10 µM Asc-1 inhibitor (BMS-466442) or DMSO as control. After 2 days, when cells reached 100% confluency, differentiation was induced with Asc-1 inhibitor or DMSO. Relative gene expression of Ucp1 (n = 3; one-way ANOVA performed with log transformed data), Ppary (n = 3), Pat2 (n = 3), Prdm16 (n = 3), and Pgc1α (n = 3). e Intracellular D-serine levels of subconfluent and confluent preadipocytes (n = 2). f Srr was knocked down using DsiSrr in proliferating shAsc-1 preadipocytes and differentiated upon confluency. Ucp1 and Ppary mRNA levels of shAsc-1 cells at day of induction (preadip) or cells differentiated following the regular differentiation protocol, including rosiglitazone (adip + Rosi). A scrambled DsiRNA was used as negative Control (Control) for knockdown (n = 4). Statistics were calculated using two-tailed t test (a) or ordinary one-way or two-way (f) ANOVA with Tukey’s or Sidak’s multiple comparison post hoc test. Data are shown as mean ± SEM.