Nbs1-S343A mutant sensitizes BCR/ABL-M07e cells to MMC treatment. (A) M07e (P) and B/A-M07e (B/A) cells were infected with empty pMig retroviral construct or those containing Nbs1-wt or Nbs1-S343A mutant. GFP⁺ cells were sorted and cultured in the presence of GM-CSF, and phosphorylation of Nbs1 on S343 was verified by Western blot analysis (pNbs1). (B) B/A-GFP empty and B/A-GFP Nbs1-S343A cells were then treated or not with 0.125 μg/mL of MMC for the indicated time periods in the presence of GM-CSF. γ-H2AX nuclear foci were detected by immunofluorescence. Percentage of highly positive living cells (> 20 foci/cell) was calculated (top panel) and the number of foci per cell was counted (bottom panel). (C) Cells were treated with 0.125 μg/mL MMC and their survival was determined by trypan blue exclusion assay. (D-F) Cells were plated in methylcellulose in the presence of GM-CSF and the indicated concentrations of MMC, HU, or Cis. Colonies were counted after 7 days. Results in panels B-F represent mean (± SD) from 3 different experiments. *P < .05 (B), *P < .004 (C), *P < .007 (D), *P < .05 (E), and *P < .03 (F) in comparison to B/A-GFP empty and B/A-GFP Nbs1-wt (if present).