Quantitative proteomics of U2O2 cells in asynchronous or synchronized in late mitosis

U2OS cells were either asynchronous or synchronized in late mitosis (anaphase/telophase) prior to irradiation. Asynchronous cells were grown to 75–80% confluence, irradiated with 4 Gy, and collected 5–10 minutes later. For synchronized samples, cells were subjected to a thymidine block, released into S phase, and then arrested in prometaphase with nocodazole. Mitotic cells were collected by shake-off, washed, and incubated in fresh medium for 35–40 minutes to allow progression into anaphase/telophase before irradiation with 4 Gy. Cell pellets were snap-frozen in liquid nitrogen for proteomics.