A549 human lung epithelial cells

DU145 human prostate epithelial cells and A549 human lung epithelial cells were acquired from American Type Culture Collection (Manassas, VA). SK-CO15 human colonic epithelial cells were provided by Dr. Enrique Rodriguez-Boulan (Cornell University). DU145 cells were cultured in RPMI medium supplemented with 10% Fetal Bovine Serum, 15% HEPES, pyruvate, and antibiotics. A549 and SK-CO15 cells were cultured in DMEM/F12 and DMEM medium, respectively, supplemented with 10% Fetal Bovine Serum and antibiotics. The cells were grown in T75 flasks (BD Biosciences), and were seeded on collagen-coated coverslips or 6-well plastic plates for immunolabeling and biochemical experiments, respectively.

A549 human lung epithelial cells and MLE 12 SV40 transformed mouse epithelial cells were obtained from the American Type Culture Collection (Rockville, MD). Both of these cell lines have type II alveolar epithelial cell characteristics. A549 cells have an intact p53-dependent G1 checkpoint. MLE 12 cells express the SV40 large T antigen, which binds to p53 leading to uncontrolled cellular proliferation and disrupts the p53 mediated G1 checkpoint [30 (link)]. Cells were cultured in DMEM/F-12, 50/50, (Cell Gro, Manassas, VA) supplemented with 10% fetal bovine serum, 50 U penicillin/ml, and 50μg/ml streptomycin in a 5% CO₂/95% air atmosphere at 37°C. Caffeine was purchased from Sigma Aldrich (St. Louis, MO, USA) and varying concentrations of Caffeine (0.05, 0.1 and 1 mM) were prepared in 1× Dulbecco’s Phosphate-Buffered Saline (Cell Gro, Manassas, VA, USA). We used 0.05 mM (≅10 mg/kg) and 0.1 mM (≅20 mg/kg) concentration to model the dose ranges used clinically in premature neonates. All cells were routinely passaged every 3 days.