Fig. 4

Fibroblast to myofibroblast transformation is inhibited by pirfenidone and rapamycin treatment. α-SMA marker was studied in primary lung fibroblasts from IPF patients treated with TGF−β (5 ng/ml), pirfenidone (1 mg/ml) and rapamycin (1 μg/ml) for 72 h. a Western blot analysis in cell lysates. Graphs represent means ± SEM of target/control ratios obtained by densitometric analysis of each experiment results. b The analysis of α-SMA gene expression after 72 h treatment showed differences between samples treated with TGF−β in the absence or presence of pirfenidone and rapamycin. α-SMA transcript fold changes expressed as relative gene expression (RGE) measured by RT-PCR in fibroblasts from IPF patients, mean ± SEM of four different experiments normalized using four different housekeeping genes as control (β-actin, GADPH, HPRT, and RNA18s). Levels of significance: ** p < 0.01; ***p < 0.001.* indicates the comparison between samples treated with TGF−β and all the other conditions: TGF−β versus untreated samples (control); rapamycin and pirfenidone (rapa/pirfe); and samples treated with TGF−β in combination with rapamycin (TGF−β/rapa); pirfenidone (TGF−β/pirfe) or both (TGF−β/rapa/pirfe). ^#p < 0.05. # indicates the comparison between untreated cells (control) and samples treated with rapamycin and pirfenidone (rapa/pirfe). n.s statistically not significant