Cell Culture and Treatments

Human RPE cell line ARPE-19 cell was purchased from the American Type Culture Collection (ATCC), and primary human RPE cells (hRPE) were kind gifts from Dr. Hong Ouyang. Both types of cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetus bovine serum (FBS), and 100 U/ml streptomycin and penicillin at 37°C with 5% CO₂ in a humidified incubator. For glucose-deprivation treatment, ARPE-19 or hRPE cells were rinsed with phosphate buffered saline twice and then treated with glucose-free DMEM with or without different percentage of FBS. At different time points as indicated, the cells were trypsinized for cell counting or harvested for western blotting or real-time polymerase chain reaction (rt-PCR). For ER stress inducing experiments, ARPE-19 cells were incubated with 2 μg/ml brefeldin A (BFA, Selleck, #S7046) in DMEM containing 2.5% FBS for 24 h.