*2.7. Apoptosis Assay*

Apoptosis analysis was performed using a FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, Waltham, MA, USA), according to the manufacturer's instructions. Briefly, radiation-induced apoptotic cells were collected at the indicated time points and resuspended in 1× diluted binding buffer in Kit. For staining, Annexin V-FITC and PI were added to each sample, and the mixture was incubated for 5 min at room temperature in the dark. The cells were analyzed immediately using a BD FACS CANTO II flow cytometer (BD Biosciences).

## *2.8. Colony Formation Assay*

Colony formation assay was performed as previously described [17]. To test the effect of IR on cell viability, appropriately seeded cells were irradiated with different doses of radiation (0, 0.5, 1, 2, 3, or 4 Gy) and incubated continuously for 2 weeks. The colonies were stained with 1% crystal violet. Colonies containing >50 cells were scored as surviving cells.

## *2.9. Invasion and Migration Assays*

Invasion assay was performed as previously reported [18]. Briefly, the cells treated with either inhibitors or siRNAs for transfection were seeded in the upper well of a Transwell chamber (8-µm pore size) that was pre–coated with 10 mg/mL growth factorreduced Matrigel (BD Bioscience). After 72 h, non–invading cells on the upper surface of the filter were removed with a cotton swab, and the migrated cells on the lower surface of the filter were fixed and stained with a Kwik-Diff kit (Thermo Fisher Scientific, Waltham, MA, USA). Invasiveness was determined by counting cells in fields per well, and the extent of invasion was expressed as the average number of cells per microscopic field. The cells were imaged by phase contrast microscopy. For the migration assay, we used Transwell chambers with inserts that contained the same type of membrane but without the Matrigel coating.
