Pirfenidone

Cell samples were collected and stromal cells were cultured as described previously [11 (link), 12 (link)]. Briefly, an aliquot of BAL-sample or collagenase-digested lung biopsy specimen was centrifuged (300 g, 10 min) and plated at a density of approximately 40,000 cells/cm² in a medium consisting of Minimun essential medium Eagle α modification (Sigma-Aldrich, Inc, St Louis, MO, USA) supplemented with 13 % heat-inactivated fetal bovine serum (PromoCell, Heidelberg, Germany), 2 mM L-glutamine, 100 U/ml penicillin, 0.1 g/l streptomycin, 2.5 mg/l amphotericin B and 10 mM HEPES (all from Sigma-Aldrich). The cells were passaged at near-confluence and used for experiments in passages 2–5. The cells were exposed to 0.1–0.5 mM pirfenidone (Santa Cruz Biotechnology) or 0.1–0.5 μM nintedanib by adding the drug into the cell culture medium with or without serum. Pilot studies were used to select drug concentrations that were low enough not to harm the cells but high enough to cause responses. The effects of the drugs were tested also in the presence of 2–5 ng/ml transforming growth factor β1 (TGFβ1) (Sigma-Alrich) in serum-free conditions.

Pirfenidone and carboxymethylcellulose were purchased from Santa Cruz Biotechnology, USA. HPFs were divided into three groups as follows: Pirfenidone group (PFD, treated with PFD at IC₅₀), blank control group (BC, without treatment) and negative control group (NC, treated with carboxymethylcellulose solution). MTT Cell Proliferation Kit was used to examine cell proliferation of HPFs (Abcam, Cambridge, MA, USA). All the procedures were performed following the instructions of the manufacturer. After cultured in 96-well plates for 24 hours in the incubator, HPFs were respectively treated with 0, 0.01, 0.1, 0.2, 0.3, 0.5 and 1 mg/ml Pirfenidone (PFD) for 6, 12, 24, 48 and 72 hours. The absorbance of optical density (OD) at 570 nm in each well was read in a microplate reader (Bio-Rad, Munich, Germany). The inhibition ratio of cell growth (IR) was calculated via the following formula: (MTT OD value of control-MTT OD value of PFD treated cells)/MTT OD value of control] × 100%. The values of IC₅₀ from HPFs were determined. Six replicates were performed for each concentration.