Fig. 6

Inhibitory effect of pirfenidone and rapamycin on tenascin-c and fibronectin gene and protein expression in bronchial epithelial human cells. A549 cells were incubated with TGF−β (5 ng/ml), pirfenidone (1 mg/ml) and rapamycin (1 μg/ml). Protein and gene expression were determined after the 72 h incubation period. a Representative immune blot showing the expression of tenascin-c in A549 cells. The bar chart summarizes the densitometric analysis of total protein expression normalized to β-actin. b Analysis of tenascin-c gene expression normalized with four different housekeeping genes (β-actin, GADPH, HPRT, and RNA18s). The bars represent the mean values ± SEM of the transcript fold changes expressed as relative gene expression (RGE) in at least three different experiments. c Fibronectin protein expression measured in cell lysates by western blot in at least three different experiments. The bar chart represents the densitometric analysis using α-tubulin as loading control. d Fibronectin gene expression was normalized using four different housekeeping genes as control (β-actin, GADPH, HPRT, and RNA18s). Bars describe the mean values ± SEM of the transcript fold changes, expressed as relative gene expression (RGE) between control and samples treated with TGF−β, pirfenidone and rapamycin. Level 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