Cell Cycle and Apoptosis Analysis

Flow cytometry analysis was performed as previously described.22 (link) For cell cycle analysis, the cells were trypsinized and washed into single-cell suspensions and fixed with ice-cold 70% ethanol at −20°C overnight. The fixed cells were then subsequently stained with 20 mg/mL propidium iodide (PI) staining buffer (containing 1% Triton X-100 and 100 mg/mL RNase A) for 30 mins. DNA content was assessed using a FACSCalibur unit (Becton Dickinson, Franklin Lakes, NJ, USA) equipped with ModFit LT v2.0 software. To analyze apoptosis, cultured cells were harvested by trypsinization and were washed with PBS. Cells (1×10⁶) from each sample were processed for annexin V-fluorescein isothiocyanate (FITC)/PI apoptosis detection according to the manufacturer’s instructions (Becton Dickinson, Franklin Lakes, NJ, USA). All the experiments were repeated at least three times.