Fig. 5.

TGF-β-induced SMC differentiation of hES-MCs was Smad dependent. A: TGF-β receptor expression in hES-MCs. hES-MCs were treated with vehicle (0) or 1 ng/ml TGF-β for various time points as indicated. TGF-β type I (TβRI) and type II (TβRII) receptors were detected by RT-PCR. Cyclophilin was an internal control. B: Smad2 and Smad3 activation in TGF-β-treated hES-MCs. hES-MCs were treated with vehicle (0) or 1 ng/ml TGF-β for the times indicated. Western blotting was performed to detect Smad2 and Smad3 expression and phosphorylation. C: Smad2 knockdown by Smad2 shRNA (shS2) was detected by Western blotting. Ctrl, control. D: Smad2 knockdown by shS2 blocked TGF-β-induced ACTA2 expression, as examined by immunostaining. GFP shRNA (shGFP) served as a control. Arrowheads indicate that the cells in which Smad2 was not knocked down expressed ACTA2. Photographs were taken at the same area for DAPI (blue), Smad2 (red), and ACTA2 (green). E: Smad3 knockdown by Smad3 shRNA (shS3) was detected by Western blotting. F: Smad3 knockdown by shS3 blocked TGF-β-induced ACTA2 expression, as examined by immunostaining. GFP shRNA served as a control. Photographs were taken at the same area for DAPI (blue), Smad3 (red), and ACTA2 (green).