Effect of nintedanib on TGF-β-induced secretion (panel A, B) and deposition (panel C) of collagens by primary human lung IPF fibroblasts (panel A, C) and by non-fibrotic control cells (B). Primary human lung fibroblasts were pre-incubated for 30 minutes with nintedanib (0.001, 0.01, 0.1, and 1 μM), before adding TGF-β (5 ng/ml) for 48 hours. Collagen secretion and deposition were quantitated by the Sircol™ Assay, and values are presented as mean ± SEM of independent experiments performed in 4 different cell lines, expressed as percentage of control (0.1% FCS) which was set to 100%. Each experiment was performed at least in duplicates. *p < 0.05. (D) Representative immuno-blots showing the expression of total and phosphorylated ERK1/2 and c-Abl in primary human lung IPF fibroblasts. Fibroblasts were starved for 24 hours. Before stimulation with TGF-β1 (5 ng/ml) for 5 minutes, cells were pre-incubated with nintedanib (1 μM) for 30 minutes. The bar charts summarise the densitometric analysis of receptor phosphorylation normalised to α–tubulin and total receptor expression in IPF cells. Data are presented as mean ± SEM of experiment performed in three different cell lines. *p < 0.05.