Fig. 3

Collagen I and III gene expression are decreased by pirfenidone and rapamycin, while only the protein expression level of collagen III is inhibited by pirfenidone and rapamycin. Primary lung fibroblasts from IPF patients were treated with TGF−β (5 ng/ml), pirfenidone (1 mg/ml) and rapamycin (1 μg/ml) for 72 h. a Densitometric analysis expressed as mean ± SEM and a representative blot showing the unchanged effect in COL1A1 levels by pirfenidone and rapamycin. b COL1A1 gene expression measured by RT-PCR was decreased by pirfenidone and rapamycin in IPF fibroblasts. Bars represent mean ± SEM values of the transcript fold changes expressed by relative gene expression (RGE) of four different experiments. c COL3A1 protein expression decreases with the combination treatment of rapamycin and pirfenidone. Bars expressed as mean ± SEM values. Treated samples with rapamycin and pirfenidone in the absence of TGF−β show a significant decrease of the protein compare to controls. d COL3A1 gene expression showing a significant decrease when cells were treated with rapamycin and pirfenidone combination treatment. Levels of significance: *p < 0.05; ** 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 < 0.001. # indicates the comparison between untreated cells (control) and samples treated with rapamycin and pirfenidone (rapa/pirfe). n.s statistically not significant