PDGF-BB stimulates Shh expression in SMCs. (A) SMCs were treated with 10 ng/mL PDGF-BB for 6 hours. The mRNA copy number of Shh, Dhh, and Ihh in stimulated (PDGF-BB) and unstimulated (Ctrl) SMCs was estimated by RT-qPCR. (B) Relative Shh mRNA expression in SMCs stimulated with PDGF-BB (10 ng/mL) for 2, 4, or 6 hours was determined by RT-qPCR; β-actin was used as housekeeping gene. (C) Concentrations of secreted Shh protein present in 24-hour–conditioned culture media from PDGF-BB–stimulated SMCs or unstimulated (Ctrl) SMCs were quantified by enzyme-linked immunosorbent assay. (D) Relative Shh mRNA expression in SMCs stimulated with different PDGF-BB concentrations was determined by RT-qPCR; data represent means ± SD from 3 independent experiments performed in triplicate. *P < .05; ***P < .0005.