Fig. 5

Inhibition of α-SMA levels was evident after addition of Pirfenidone. CT- and DD-derived fibroblasts derived from three different patient samples (N = 3/group) of were left untreated (Ntx) or were stimulated with PFD (800 μg/ml) in the presence or absence of TGF-β₁ (10 ng/ml) in α-MEM medium containing 0.1% dialyzed FBS. Twenty-four hours later cell lysates were collected to determine the mRNA and protein expression of α-SMA by real-time RT-PCR (a & b) and Western blot analyses (c & d). Real-time RT-PCR experiments were done on three independent cultures of each of the cell types. Values are mean ± SEM of three independent studies performed in triplicate. One-way ANOVA test was used to determine the statistical significance. *p < 0.05, **p < 0.01. Western blot analyses shown here are representative images of experiments performed using three independent primary cultures of CT- and DD-cord derived fibroblasts. Proteins obtained from different patients were processed in parallel to confirm the changes that are observed are consistent. Protein accumulation was quantified by densitometry using GAPDH as a loading control (e)