Down-regulation of Nbs1-S343 phosphorylation followed by inhibition of BCR/ABL kinase sensitizes BCR/ABL-positive cells to MMC. (A) B/A-M07e (A/B) cells were infected with empty pMig retroviral construct (empty) or containing Nbs1-S343A mutant (Nbs1-S343A). GFP⁺ cells were sorted and cultured in the presence of SCF. Cells were treated (+) or not (−) with 1 μM IM for 24 hours and/or 0.5 μg/mL MMC for 12 hours in the presence of GM-CSF. Expression of pNbs1, Nbs1, BCR/ABL, and phosphotyrosine proteins (p-Tyr) was examined by Western-blot analysis. (B) Clonogenic assay was performed on cells preincubated with 1 μM IM that were then treated with 0.025 μg/mL MMC (MMC1), 0.05 μg/mL MMC (MMC2), or both (IM + MMC1, IM + MMC2) in the presence of GM-CSF. Colonies were counted 7 days later. Results represent mean percentage of colonies (± SD) in comparison to the colonies formed by untreated cells. *P < .005 in comparison to the corresponding empty cells treated with MMC + IM; *P < .05 in comparison to corresponding Nbs1-S343A cells treated with MMC or IM.