Fibroblast Cell Culture and TGF-β1 Stimulation

The normal human lung fibroblast cell line, IMR-90 (Japanese Collection of Research Bioresources, Osaka, Japan), and the IPF lung fibroblast cell line, LL97A (American Type Culture Collection, Manassas, VA, USA), were cultured in Eagle’s Minimum Essential Medium (MEM) with Earle’s salt (GIBCO) supplemented with 10 % fetal bovine serum, 100 U/ml of penicillin, 100 μg/ml of streptomycin and 0.1 mM nonessential amino acids. The African green monkey fibroblast line, COS1 (American Type Culture Collection, Manassas, VA, USA), was also cultured as described previously [12 (link)]. IMR-90 and LL97A cells were plated in six-well plates and stimulated for 24 h with recombinant TGF-β1 (5 ng/ml) (R&D Systems, Inc., Minneapolis, USA). We performed a Real-Time qPCR analysis to examine the time dependency of the expression profiles of the WNT ligands and COL1A1 by stimulation with TGF-β1, and performed the Sircol collagen assay to quantify the total collagens. In addition, whole-cell lysates were prepared and subjected to Western blot analyses.