Effect of nintedanib on receptor expression and phosphorylation. (A) Fibroblasts derived from patients with IPF were treated with increasing concentrations of nintedanib (0.01, 0.1, and 1 μM) for 24 hours and cell viability was analysed by trypan blue exclusion staining. (B) Representative immuno-blots showing the expression of total PDGF-receptor (PDGFR), VEGF-receptor (VEGFR) and FGF-receptor (FGFR), in primary human lung IPF fibroblasts and non-fibrotic control cells. The bar charts summarise the densitometric analysis of total receptor expression normalised to GAPDH in IPF and control cells. Data are presented as mean ± SEM of experiment performed in three different cell lines. *p < 0.05. (C) Representative immuno-blots showing total and phosphorylated PDGFR, VEGFR, and FGFR in primary human lung IPF fibroblasts. Fibroblasts were starved for 24 hours. Before stimulation with PDGF-BB, bFGF, or VEGF (all 10 ng/ml) for 30 minutes, cells were pre-incubated with nintedanib (400 nM) for 30 minutes. The bar charts summarise the densitometric analysis of receptor phosphorylation normalised to total receptor expression in IPF cells. Data are presented as mean ± SEM of experiment performed in three different cell lines. *p < 0.05.