Apoptosis was detected using an Annexin V- fluorescein isothiocyanate (FITC) apoptosis kit (Nanjing Keygen Biotech Co., Ltd., China), according to the manufacturer's protocol. Cells were maintained under stress conditions at 37°C for 24 h (glucose and amino acid starvation) prior to measuring the rate of apoptosis. Briefly, cells were collected and stained with Annexin V-FITC and propidium iodide (PI) (1 µl Annexin V-FITC and PI in 100 ml cell suspension) for 15 min at room temperature, and the cell apoptotic rate was measured by flow cytometry (BD FACSVerse 4 color Flow Cytometer; BD Biosciences).
For cell cycle analysis, cells were maintained in serum-free medium for 24 h, after which, they were collected and fixed with 70% ethanol overnight at 4°C. Subsequently, cells were washed three times with 1X PBS, stained with PI (1 µl PI in 100 ml cell suspension) for 15 min at room temperature and the cell cycle progression was measured by flow cytometry (BD FACSVerse 4 color Flow Cytometer; BD Biosciences).
For cell cycle analysis, cells were maintained in serum-free medium for 24 h, after which, they were collected and fixed with 70% ethanol overnight at 4°C. Subsequently, cells were washed three times with 1X PBS, stained with PI (1 µl PI in 100 ml cell suspension) for 15 min at room temperature and the cell cycle progression was measured by flow cytometry (BD FACSVerse 4 color Flow Cytometer; BD Biosciences).