Fig. 4

Effects of pirfenidone on TGF-β1-mediated fibrotic regulators in lung fibroblasts. Subconfluent HFL-1 cells were cultured in SF-DMEM for 24 h and then incubated in the presence or absence of TGF-β1 (0.25 ng/mL) and pirfenidone (100 ng/mL). (a) CTHRC1 and FHL2 expression in HFL-1 cells was determined using fluorescence-immunohistochemistry with primary antibodies against CTHRC1 or FHL2, followed by incubation with secondary antibodies labeled with Alexa Fluor 488 goat anti-rabbit IgG (green). The scale bar indicates 50 μm. (b) Western blot analysis to determine the effects of pirfenidone on targets related to TGF-β1-mediated fibrotic processes, including CTHRC1 (30 kDa), FHL2 (30 kDa), α-SMA (42 kDa), fibronectin (250 kDa), BMP4 (47 kDa), Gremlin1 (25 kDa), and β-actin (42 kDa). The vertical axis shows the relative intensities of (c) CTHRC1, (d) FHL2, (e) α-SMA, (f) fibronectin, (g) BMP4, and (h) Gremlin1 versus β-actin; the horizontal axis shows the conditions. Values represent means ± SEMs of three to five separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001