HCT116 cells growing in chamber slides were treated with 15 μM 5-FU (or vehicle) for 18 hours and 250 nM selumetinib (or vehicle) for 2 hours prior to irradiation with 4 Gy. Following irradiation, cells were washed with PBS, and media containing 250 nM selumetinib (or vehicle) was added. A) At the indicated time-points, cells were fixed and subjected to immunocytochemistry for γH2AX. The percentage of cells with greater than 50 foci per cell were determined for each treatment condition. The percentage of cells with >50 foci per cell was significantly increased at 24 hours after 4 Gy in cells treated with 5-FU+selumetinib compared to vehicle treated cells. B) Representative images of γH2AX foci (γH2AX labeled with red, nuclei with blue). Columns, mean from 3 independent experiments; bars, standard deviation; *, p<0.05 (selumetinib + 5-FU + 4 Gy vs. vehicle + 4 Gy and selumetinib + 4 Gy).