Clonogenic survival assay

To measure cellular sensitivity to cytotoxic therapy such radiation and chemical treatments, clonogenic survival assay was performed according to an established protocol (44). Briefly, the cells were treated with mitomycin C at different concentration for 72 hours or irradiated with different doses of X rays. They were then plated in triplicate 10 cm dishes at different numbers according to mitomycin C concentration or radiation doses so that there would be 50 200 colonies form eventually in each well. After colonies are clearly visible (about 11 to 14 days), cells were fixed and stained with 0.5% crystal violet. Colony numbers are then counted and the surviving fractions were calculated according to the number of initially plated cell numbers and the clonogenicity (calculated as % of colonies formed from all inoculated cells) of untreated control cells. In some cases, cells were treated with caspase 3 inhibitor Z DEVD FMK at 15 μM for 4 hours and subsequently exposed to different concentrations of mitomycin C or different doses of X rays, and then incubated in the culture medium containing 1μM caspase 3 inhibitor Z DEVD FMK or vehicle for 14 days.