MMC-induced G₂/M checkpoint is partially obviated by SLFN11 disruption. (A) Cell cycle profile. FANCD2^−/− and FANCD2^−/−SLFN11^−/− cells were treated with 1 ng/mL MMC for 48 hours and pulse-labeled with BrdU. Cells were then fixed in 70% ethanol, stained with either PI alone (i) or with anti-BrdU and PI (ii), and analyzed using a FACS Canto II flow cytometer (Becton-Dickinson). (B) CHK1 activation by Western blotting. CHK1 phosphorylation on Serine 345 was examined at the indicated time points during 20 ng/mL MMC exposure in wild type, SLFN11^−/−, FANCD2^−/−, and FANCD2^−/−SLFN11^−/− HAP1 cells. The loading was equalized based on the amount of protein, and the weaker tubulin bands in FANCD2^−/− cells were likely caused by the toxicity of MMC treatment. (C) Chromosome breakage analysis. Cells were subjected to 40 ng/mL MMC exposure for 24 hours. Each dot represents the number of chromosomal breaks in a single metaphase, and means ± SEM are shown. More than 50 metaphases were scored in each condition. Representative images of metaphases after MMC treatment are shown. P values were calculated by unpaired, 2-tailed Student t test.