Fig. 7

Pirfenidone and rapamycin combined treatment inhibit the expression of collagen type I and III induced by TGF−β in alveolar human epithelial cells. A549 cells were incubated with TGF−β (5 ng/ml), pirfenidone (1 mg/ml) and rapamycin (1 μg/ml) for 72 h. a and c The expression of COL1A1 and COL3A1 were measured by western blot. Representative immunoblot analysis of at least three different experiments normalized by α-tubulin. The combination of rapamycin and pirfenidone treatment inhibits the protein expression of collagen type III. Gene expression b and d were analyzed in A549 cells after the drug treatment by RT-PCR. Reported values are means ± SEM of the transcript fold changes expressed as relative gene expression (RGE) normalized by four housekeeping genes and calculated on the level of untreated (control) cells. Only collagen type III gene expression is decreased by the treatment of rapamycin and pirfenidone. Levels of significance; *p < 0.05; **p < 0.01 * 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). n.s statistically not significant