Cell culture
A549 cells (human lung epithelial cell line) or LL29 cells (lung fibroblasts from an IPF patient) were cultured in DMEM supplemented with 10% FBS or Ham’s F-12K (Kaighn’s Modification) medium supplemented with 15% FBS, respectively, in a humidified atmosphere of 95% air with 5% CO2 at 37°C.
Viable cells were measured as previously described (Nakano et al., 2020; K. I. Tanaka et al., 2019). Briefly, dissociated A549 or LL29 cells were added to 96-well culture plates at a concentration of 1 × 104 cells per well in 200 µl of culture medium. After a 24-h incubation, cells were treated with various reagents added to the medium. After 24 h, the percentage of viable cells was quantified using CellTiter-Glo® 2.0 (Promega Corporation, Madison, WI, USA). The cytotoxicity in LL29 cells after eperisone addition was measured every hour using CellTox™ Green Dye (Promega Corporation, Madison, WI, USA) and a microplate reader (Tecan, Kawasaki, Japan; excitation: 485 nm, emission: 530 nm).