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Fig. 7. The role of the TGF-ß-dependent pathway in SPC-induced SMC differentiation and the late activation of Smad2. (A) Serum-starved hATSCs were pretreated with 10 µM SB-431542 or vehicle, and then treated with 2 µM D-erythro-SPC or 2 ng/ml TGF-ß3 for 4 days. The expression levels of -SMA and actin, and the phosphorylation levels of ERK and Smad2 were determined by western blotting. Representative data from three independent experiments are shown. (B) hATSCs were exposed to 2 µM D-erythro-SPC for 2 days in the absence or presence of 10 µM U0126, and then the conditioned media were subjected to ELISA for quantification of TGF-ß1 protein. Data are shown as mean ± s.e.m. (n=4) and representatives of three independent experiments. ^*P<0.05. (C) Serum-starved hATSCs were exposed to 2 µM D-erythro-SPC for the indicated time periods, and TGF-ß1 and GAPDH mRNAs were quantified by semi-quantitative RT-PCR. Representative data from three independent experiments are shown. (D) Serum-starved hATSCs were treated with 2 µM D-erythro-SPC in the absence or presence of 10 µM U0126 for 6 hours. The mRNA levels of TGF-ß1 and GAPDH were determined by semi-quantitative RT-PCR. (E) The relative level of TGF-ß1 was normalized to those of GAPDH. The data are shown as mean ± s.e.m. of triplicate determinations. ^*P<0.01. (F) Serum-starved hATSCs were treated with 2 µM D-erythro-SPC, 2 ng/ml TGF-ß1, or 2 ng/ml TGF-ß3 together with anti-TGF-ß1 neutralizing antibody (0.2 µg/ml) for 4 days. The expression levels of -SMA and actin, and the phosphorylation level of p-Smad2 were determined by western blotting. Representative data from three independent experiments are

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