Cell cycle analysis

Cell cycle phases were analyzed by measuring the DNA fragment staining with propidium iodide (PI, Sigma-Aldrich Co.) and used according to the manufacturer’s instruction. A549 cells in the exponential growth phase were plated in six-well plates, divided, and treated as noted earlier. The cells were harvested and centrifuged at 24, 48, and 72 hours after exposure to10 Gy irradiation. Cell pellets were washed twice with phosphate-buffered saline. The cells were fixed with 70% ethanol at 4°C overnight and incubated with 200 μL DNAse-free RNAse (Sigma-Aldrich Co.) for 30 minutes at room temperature in the dark. Then, the cells were analyzed immediately by flow cytometry with a FACScanTM system using the CellQuestTM software (Version 3.3) (BD Biosciences, San Jose, CA, USA).