Cell Cycle Analysis in Cancer Cells

Cell cycle was determined as previously described [25 (link)] with a slight modification. Briefly, HCT116 and H1299 cells (3 × 10⁵ cells/well) were seeded in 10-cm culture dishes (Corning Inc.). After 24 h, cells were synchronized for another 24 h in serum free media. Cells were then treated with PLE at 350 µg/ml in serum complete media for 24 h (in the case of H1299) or 72 h (in the case of HCT116). Adherent cells were detached by brief trypsinization (0.25% trypsin-EDTA; Sigma-Aldrich). Cell pellets were washed with ice-cold PBS and then re-suspended in 70% ethanol overnight for fixation. After centrifugation at 3,000 × g, the supernatant was removed, and cells were incubated with PBS containing 1 µg/ml RNAse (Sigma-Aldrich) and 50 µg/ml propidium iodine (Sigma-Aldrich) for 20 min at room temperature. Single-cell suspension was generated by gentle pipetting. Cell cycle analysis was performed using a flow cytometer (FACS Calibur-S System, BD Biosciences, Heidelberg, Germany), and data were processed using Cell Quest Pro software (BD Biosciences).