Figure 4
Expression of MiTF-WT enabled a temporary G1 arrest for improving cell survival after UVR. A, A375 cells were transfected with GFP, MiTF-WT or MiTF-S73A and then exposed to UVC at 3 mJ/cm2, and fixed 8 hours later for FACS analysis after Propidium Iodide staining. B, sub-G1 population of cells treated in A were calculated by FACS analysis and graphed 24 hours after UVC treatment. C, cells in A were seeded and exposed to UVC, then incubated for colony formation assay. Colonies formed 2 weeks after were counted, normalized to that in GFP-expressing cells and graphed. D, A375, WM3211, 1205Lu, Malme-3 M, SK-Mel-28 and c83-2C cells were exposed to UVC at 3 mJ/cm2, and cells were then collected 24 hours later for FACS analysis. E, Percentage of cell death before and after UVC were calculated and graphed F, knockdown of MiTF decreased cell survival after UVC. MiTF was knocked down by Mish1 and Mish2 shRNA (see Fig 5E) and exposed to 3 mJ/cm2 of UVC. Colony formation was analyzed about 2 weeks post-radiation.