Fibroblast Culture and Proliferation Assay

Human lung fibroblasts MRC5 (CCL-171; ATCC, Wesel, Germany) were cultured in Ham’s F12 medium (Life technologies, Carlsbad, United States) supplemented with 10% (v/v) fetal bovine serum (FBS, PAA Laboratories, Pasching, Austria), 2 mM l-glutamine (Life Technologies #35050–061), 100 U/mL penicillin/streptomycin, and 1% amphotericin B (1x, Gibco). CCL-206 mouse lung fibroblasts ([MLg2908, CCL206], ATCC, Wesel, Germany) were cultured in DMEM/F12 medium supplemented with 10% FBS, penicillin/streptomycin (100 U/mL), glutamine (1%) and amphotericin B. Cells were incubated at 37°C, 5% CO₂ humidified environment. MRC5 or CCL206 fibroblasts were starved once grown to 80% confluence in the 6-well culture plates. The starvation medium contains the same components as culture medium described above, but with only 0.5% FBS. After 24 h starvation, cells were then incubated with either vehicle, TGF-β1 and/or investigational compounds (Table 1) in serum deprivation medium for 48 h. MRC5 fibroblasts were collected for gene expression analysis. CCL206 fibroblasts were washed three times with warm PBS and proliferation-inactivated by incubation in mitomycin C (10 μg/ml, Sigma #M4287) for 2 h, followed by three washes in warm PBS and trypsinization prior to mixing with epithelial cells, as described previously (Ng-Blichfeldt et al., 2018 (link); Ng-Blichfeldt et al., 2019 (link)).