Figure 3.

Blockade of TGFβ signaling pathway markedly improved adipogenesis in Om ASCs. A: Paired Om and Abdsc ASCs were differentiated in the absence (−) or presence (+) of SB (5 μmol/L), and differentiation (Diff.) degree (ATGL protein) was measured on day 14. Each symbol represents ASCs derived from a different donor. Scale bars, 100 μm. B: Representative images of differentiated cells. Effects of depot and SB on adipogenesis were determined by two-way ANOVA followed by paired t tests (*P < 0.05, **P < 0.01; n = 5). C: Om ASCs were transfected with control, SMAD2, SMAD3, both SMAD2 and 3 (SMAD2/3), or SMAD4 siRNA, and KD levels were confirmed at confluence with immunoblotting. D: Cells were differentiated and PPARγ mRNA levels were measured as an adipocyte marker; dotted line represents the average value for paired Abdsc ASCs, differentiated in the control condition. Data were first analyzed by RM-ANOVA followed by Dunnett test to compare experimental values with the control (*P < 0.05 and **P < 0.01) and a paired t test to compare SMAD2/3 KD with SMAD2 KD alone (#P < 0.05); n = 6. E: Paired Om and Abdsc ASCs were transfected with control or activin A (INHBA) siRNA, and expression levels of activin A, phosphorylated SMAD2 (PSMAD) and total SMAD2 (TSMAD2), and HSP90 were measured at confluence. F: Differentiation degree was evaluated by measuring ATGL protein levels on day 14. Each symbol represents ASCs derived from a different subject. G: Representative images of differentiated cells are presented. Depot and siRNA effects on adipogenesis were determined by two-way ANOVA followed by paired t tests (*P < 0.05; n = 5).