Cell cycle assay by flow cytometry

For the cell cycle analysis, cells (3×10⁵) were seeded in 10-cm plates and incubated for 24 h. The cells were exposed to PEM (0–110 nM) with fresh medium for 96 h at 37°C. Cells were trypsinized and washed, then resuspended in 75% ice-cold ethanol (75%) overnight at 4°C. This was followed by washing with 1X PBS and centrifugation; the cell pellets were gently dispersed with 300 µl of 1X PBS with 30 µl of RNase A (10 mg/ml) for 30 min. Propidium iodide (PI) (10 µl; 500 µg/ml) and 300 µl of 1X PBS were added followed by another 30 min incubation at room temperature. Subsequently the cells were filtered by a nylon mesh (40 µm), and then analyzed with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).