Bovine pituitary extract

RLE-6TN cells (lot no. 59111690), a rat lung epithelial cell line with characteristics of alveolar type II cells, were purchased from the American Type Culture Collection (Rockville, MD, USA). All experiments using this cell line were performed within 4 months after resuscitation. RLE-6TN cells were grown in Ham's F12 medium containing 2 mM L-glutamine (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 10 μg/ml bovine pituitary extract (PromoCell, Heidelberg, Germany), 5 μg/ml insulin (Gibco), 2.5 ng/ml insulin-like growth factor (Sigma-Aldrich, St. Louis, MO, USA), 1.25 μg/ml transferrin (Gibco), and 2.5 ng/ml epidermal growth factor (Sigma-Aldrich, St. Louis, MO, USA) as we described previously (Hagiyama et al., 2015 (link)). H₂O₂ (Wako Pure Chemical Industries, Osaka, Japan) was added to the semiconfluent culture of RLE-6TN cells at a concentration of 440 μM, according to the procedures published previously (Kim et al., 2014 (link)). After 4 or 8 h, H₂O₂ was removed by medium replacement, and the cultures were continued for 2 or 3 days. In another experiment, elastase (porcine pancreas; Worthington, Lakewood, NJ, USA) was added at a concentration of 1 unit/ml to the semiconfluent culture of RLE-6TN cells grown in the Ham's F12 medium same as described above except lacking fetal bovine serum. After 1 h, cells were subjected to Western blot analyses.