Figure 1.

Involvement of Smad2, EGF-R, and Alk5 in phenotypic conversion. Confluent monolayers of young dermal fibroblasts were transfected with Smad2 siRNA or scramble oligonucleotide control (scramble) for 48 hours. A: Total mRNA was extracted and Smad2 mRNA expression was assessed by RT-Q-PCR. B: In parallel experiments, after transfection the medium was replaced with serum-free medium for a further 48 hours, before addition of serum-free medium alone (white bars) or serum-free medium containing 10 ng/ml TGF-β1 (black bars) for a further 72 hours, and α-SMA expression was assessed by RT-Q-PCR. C: Confluent monolayers of young dermal fibroblasts were growth-arrested in serum-free medium for 48 hours. Serum-free medium alone or serum-free medium containing 10 ng/ml TGF-β1 alone or in combination with either the EGF-R inhibitor AG1478 (10 μmol/L) or the Alk5-specific inhibitor SB431542 (10 μmol/L) was added to cells for a further 72 hours. Total mRNA was extracted, and α-SMA expression was assessed by RT-Q-PCR. Ribosomal RNA expression was used as an endogenous control, and gene expression was assessed relative to scramble (A), unstimulated-scrambled (B), and control unstimulated (C) samples. The comparative C_T method was used for relative quantification of gene expression, and the results represent the mean ± SE of six individual experiments using cells isolated from two different donors. Statistical analysis was performed by Student's t-test: *P < 0.05.