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. 2013 May;54(5):1385–1396. doi: 10.1194/jlr.M035576

Fig. 7.

Fig. 7.

TGF-β acts upstream of STAT3 in the butein mediated anti-adipogenesis. A: Transient transfection of 3T3-L1 cells with two independent combinations of SMAD2 #1 and SMAD3 #1 (15 μM of each, si #1) or SMAD2 #2 and SMAD3 #2 (15 μM of each, si #2) siRNAs reduced SMAD2 and SMAD3 expression. B: Silencing TGF-β signaling increased adipocyte differentiation. Transient transfection of 3T3-L1 cells with two independent combinations of SMAD2/3 siRNAs (si #1 or si #2) promoted lipid accumulation as assessed by Oil Red O staining. C: Knockdown of TGF-β signaling attenuated butein's inhibitory effects on expression of STAT3 target genes (KLF5, P53, C/EBPδ, and CycD1) and butein's stimulatory effects on expression of TGF-β target genes (SMAD7 and PAI-1). Expression of STAT3 and TGF-β target genes in nonspecific control and SMAD2/3 siRNA-transfected cells treated with DMSO or butein (10 μM) was measured by real-time PCR. D: Control (ctrl) and SMAD2/3 knockdown (KD, si #1) cells were cultured in the presence of DMSO or butein (10 μM) for the various time points indicated and expression profiles of TGF-β and STAT3 target genes were measured. Data shown represent the mean ± SEM from three independent experiments. Statistical significance was determined relative to a control by the Student's t-test (*P < 0.05; **P < 0.005; ***P < 0.0005). Norm, normalized.