Hyperoxia-Induced Lung Epithelial Cell Stress

Mouse lung epithelial (MLE)-12 cells were cultured in DMEM/F-12 medium (Invitrogen) supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (10 μg/mL) at 37°C and 5% CO₂ in humidified air as described previously (34 (link)). These cells were exposed to hyperoxia (95% O₂ and 5% CO₂) for 72 hours. The cells were pretreated with vehicle, 4-PBA (0.3 mM), or claudin-4 siRNA (25 nM). The dose of 25 nM claudin-4 siRNA used in MLE-12 cell was determined by Western blot (Supplementary Figure 2). For claudin-4 siRNA transfection, MLE-12 cells were incubated for 24 hours and transfected with claudin-4 siRNA, using DharmaFECT™ 1 siRNA Transfection Reagent (Dharmacon Inc. Chicago, IL, USA). MLE-12 cells with non-targeting control siRNA treatment were used as negative controls. After 24 hours, the culture medium was replaced with the recommended medium. Forty-eight hours after claudin-4 siRNA treatment, MLE-12 cells were exposed to hyperoxia for 72 hours. Next, cells were lysed for protein extraction subjected to the western blot procedure. The supernatant was collected and assayed for KC using a mouse KC ELISA kit (R&D, Inc.). The experiments were performed in triplicates.