Clonogenic assay to measure cell survival

Cell survival was determined using a standard clonogenic assay. Cells with or without SMAD4 knockdown were trypsinized to generate a single-cell suspension and seeded into 6-well plates in triplicate. After incubation for 16 h, 800 cells were treated with IR (0–6 Gy). After 7–10 incubation, the colonies were fixed with 4% paraformaldehyde and then stained with 0.5% crystal violet. After counting the number of colonies (containing more than 50 cells), the surviving fraction was calculated.