HES1 is required for MMC resistance. (A) HeLa cells transfected with 3 different Stealth RNAi against HES1 or scrambled sequences were treated with various concentration of MMC. The cells were incubated for 5 days before counting. (B) HES1 is required for FANCD2 monoubiquitination. Ubiquitination analysis of FANCD2 in HeLa cells that were depleted of HES1 by either Stealth RNAi against HES1, HES1 scrambled sequences (negative control), or the γ-secretase inhibitor L685,458. (C) FANCD2 foci formation in HeLa cells. HeLa cells depleted of HES1 using either Stealth RNAi against HES1, HES1 scrambled sequences (negative control), or the γ-secretase inhibitor L685,458 were treated with 120 ng/mL MMC and subjected to immunofluorescence analysis 16 hours later. White boxes indicate selected cells (middle panel) shown at higher magnification in the bottom panel. (D) Reduction in the number of DNA damage–induced FANCD2-containing foci cells following HES1 depletion. Cells containing more than 5 bright foci were scored as FANCD2-positive cells. Mean (± SD) of 3 separate experiments each from 100 cells. *P < .01; **P < .05.