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. 2019 Dec 12;9:18925. doi: 10.1038/s41598-019-55176-w

Figure 2.

Figure 2

Mediators implicated in pulmonary organization induce myofibroblast differentiation of normal and IPF fibroblasts. Serum starved human fibroblasts were treated with various mediators to induce myofibroblast differentiation (TGF-β, FXa, thrombin (THB), plasmin (PLN) and uPA; see Materials and Methods). Cell lysates and conditioned medias, collected after 48 h, were then resolved by SDS-PAGE and western blotted for α-SMA, total GSK-3β, tyrosine 216 phosphorylated GSK-3β (pTyr-GSK-3β) and collagen 1 (Col-1), in NF (A) and IPF cells (C). β-actin was the loading control. α-SMA and collagen 1 expression were then quantified by densitometric analyses. Plotted data are the mean ± SEM of n = 3 independent experiments. Collagen was most prominently induced by TGF-β and FXa. Images are representative of three independent experiments. NF (B) and IPF (D) cells were treated PBS, TGF-β, Xa, thrombin, plasmin and uPA for 24 h incubation. RNA was then collected, and qPCR analyses were then performed for α-SMA and collagen 1 expression. GAPDH was the loading control. Plotted data are the mean ± SEM of n = 3–4 independent experiments.