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. 2020 Jul 2;10:10900. doi: 10.1038/s41598-020-67904-8

Figure 1.

Figure 1

Inhibitory effects of pirfenidone on the activation of primary cultured fibroblasts. (A) Cancer-associated fibroblasts (CAFs) are defined as fibroblasts obtained from tumors. Lung normal fibroblasts (LNFs) are defined as fibroblasts obtained from normal lung tissue. CAFs and LNFs are treated with DMSO (control) or pirfenidone (PFD, 500 μg/mL) for 2 days. Phase contrast pictures were taken. Scale bar: 100 µm. (B) CAFs and LNFs are treated with DMSO or PFD for 24 h. Subsequently, total RNA is extracted, and real-time RT-PCR is performed to detect ACTA2, collagen I, IL-6, hyaluronan synthase 2, and VEGF mRNA. All experiments were performed three times independently with duplicates, and the results are the means ± SD. Significance was tested with the Mann–Whitney U test. (C) CAFs and LNFs are treated with DMSO or PFD for 2 days and then analyzed by Western blotting for α-SMA, collagen I, and GAPDH (loading control). All experiments were performed two times independently. Data shown are representative experiments and their quantitative values. (D) CAFs and LNFs are treated with DMSO or PFD for 2 days. The medium is changed to FBS-free medium, and TGF-β1 or IL-6 in the conditioned medium is then measured 24 h after the medium change using ELISA. All experiments were performed three times independently with duplicates, and the results are the means ± SD. Significance was tested with the Mann–Whitney U test.