Fig. 5. The role of PTX-sensitive G proteins in the SPC-induced differentiation of hATSCs to SMCs. (A) Serum-starved hATSCs were treated with 2 µM D-erythro-SPC or 2 ng/ml TGF-ß3 for the indicated time periods, and the phosphorylation level of Smad2 was determined by western blotting with anti-phospho-Smad2 antibody. Anti-actin antibody was used to confirm equal loading. (B) The densities of p-Smad2 and actin were quantified in duplicate, and the phosphorylation levels of Smad2 were normalized to total actin levels in the samples. Relative densities of p-Smad2 are shown as mean ± s.e.m. Asterisks indicate significant difference from control values (^*P<0.05, #P<0.01). (C) hATSCs were pretreated with 100 ng/ml PTX for 24 hours. The cells were then exposed to 2 µM D-erythro-SPC or 2 ng/ml TGF-ß3 for 10 minutes or 4 days, and the phosphorylation level of Smad2 was determined by western blotting with an anti-phospho-Smad2 antibody. Anti-actin antibody was used to confirm equal loading. Representative data from three independent experiments are shown. (D) Serum-starved hATSCs were pretreated with 100 ng/ml PTX for 24 hours. The cells were then exposed to 2 µM D-erythro-SPC or 2 ng/ml TGF-ß3 for 4 days, and the expression levels of -SMA and actin were determined by western blot analysis. Representative data from three independent experiments are shown. (E) The densities of -SMA and actin were quantified from the duplicate determinations, and the expression levels of -SMA were normalized to total actin levels in the samples. The data are presented as a percentage of control (mock-treated cells). ^*Significantly different from control value (P<0.05).