Transduction of TGF-β signals in HDLECs. (A) Immunoblotting of phospho-Smad2 after TGF-β or TβR-I inhibitor treatment. HDLECs were treated with TGF-β1 or with 2 kinds of TβR-I inhibitors (LY364947 or SB431542, shown as Inhib1 or Inhib2, respectively) in the presence and absence of TGF-β1 for 1 hour, and subjected to immunoblot analysis using phospho-Smad2 antibody (top panel) and Smad2/3 antibody (bottom panel). Ctrl indicates control. (B,C) Real-time PCR of Smad7 and PAI-1. HDLECs were treated as in panel A, and expression of Smad7 and PAI-1 mRNAs was determined at 1 hour and 24 hours after stimulation, respectively (***P < .001). (D) Regulation of growth of HDLECs by TGF-β and TβR-I inhibitor. HDLECs were seeded at a density of 2 × 10³ cells/well in 96-well plates, and cells were treated or not with TGF-β1 (1 ng/mL) or LY364947 (3 μM). Photographs of the cells were taken at day 2. Cell numbers were determined by WST assay in triplicate at day 2. Error bars represent standard deviations (*P < .05, **P < .01). (E,F) Autocrine TGF-β signaling in HDLECs. Expression of TGF-β1 mRNA in HDLECs treated with TGF-β1 (1 ng/mL) or TβR-I inhibitors (3 μM) as in panel A was determined by real-time PCR (E). Production of TGF-β1 protein by HDLECs treated as in panel A, but with TGF-β3 (1 ng/mL) as the stimulant, was examined in conditioned medium using an ELISA kit (F). LY364947 was used as TβR-I inhibitor (Inhib). HUVECs were used as a control. Error bars represent standard deviations (*P < .05, **P < .01, ***P < .001).