Figure 3.

Effects of S6 (a Gas6 decoy receptor) and BIBF1120 on normal and idiopathic pulmonary fibrosis (IPF) myofibroblast differentiation. (A) Cultured fibroblasts from normal and IPF lungs were examined by Western blotting for the expression of Axl, Mertk, Tyro3, and β-tubulin. Fibroblasts cultured from normal and IPF lungs were treated with S6 (4 μg/ml), BIBF1120 (300 nM), or vehicle and exposed to CpG (10 μM) for 24 or 72 hours. (B) Soluble Axl was measured in untreated fibroblast cultures from normal (n = 3) and IPF (n = 6) cells lines by ELISA. (C and D) Free Gas6 was measured by ELISA in normal (n = 3) and IPF (n = 5) fibroblast cultures treated with S6 (C) or BIBF1120 (D). (E) Western blot analysis of α-SMA and total Axl relative protein expression were quantified from normal (n = 4) and IPF (n = 5) fibroblast cultures treated with S6 and BIBF1120 in the absence or presence of CpG for 24 hours. Relative protein expression was calculated from ratios of α-SMA or total Axl/β-tubulin, and results were expressed compared with untreated samples. (F) Representative blots for α-SMA, total Axl, and β-tubulin. NL26789 and S170 are individual cell lines used in our experiments. Data are mean ± SEM. *P ≤ 0.05. α-SMA = α-smooth muscle actin; Axl = anexelekto; BIBF = BIBF1120 (nintedanib); Gas6 = growth arrest–specific 6; Mertk = MER proto-oncogene, tyrosine kinase; N.D. = not detected; sAxl = soluble Axl; Tyro3 = TYRO3 protein tyrosine kinase 3; Unt = untreated.