Cells were transfected with 30 nM of S1P1, S1P2, or S1P3 receptor siRNA or non-silencing siRNA for 24 h, then serum starved overnight. Depletion of S1P receptor isoforms was assessed by qPCR. A. Control cells were treated with 1 μM S1P or 2.5 ng/ml of TGF-β plus 0.5 μM dhS1P for 30 min. to assess Smad3 phosphorylation level. B. SSc fibroblasts were treated with S1P(s) or dhS1P (d) for 30 min. to assess Smad3 phosphorylation level. Phospho-Smad3, total Smad3, and β-actin were detected by western blotting.