Cell Line Cultivation Protocols

NCI-H441 cells (lot no. 58294188), a human lung epithelial cell line with characteristics of Clara cells, and 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) in 2010 and 2013, respectively. All experiments using these cells were performed within 4 months after resuscitation. NCI-H441 cells were grown as described in our previous report [4 (link)]. 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). COS7 and NIH3T3 cells were described previously [14 (link), 15 (link)]. COS7 cells that overexpressed CADM1 exogenously were described previously [11 (link)].