Cotreatment triggers DNA damage accumulation in MM cells. (A) Western blot analysis of γ-H2A.X in the indicated MM cell lines treated with Erw-ASNase (0.5 U/mL), Kar (3 nM), or their combination for 24 hours. GAPDH was used as loading control. (B) Immunofluorescence staining of γ-H2A.X in treated LP1 cells (left panels). The number of γH2AX foci per cell was quantified and is shown normalized to control (right panel). Magnification ×40. (C) Western blots showing DNA damage and DDR pathway deregulation of LP1 cells after drug treatment (left panels). Quantification of each signal is shown normalized to γ-tubulin as loading control (right panel). (D) HR activity of U266 after the indicated drug treatment. Cells were treated for 6 hours, nucleofected with dl1 and dl2 plasmids (250 ng each), and treated for an additional 20 hours. Next, DNA was extracted, and qPCR was performed. 2^DDCt was used to quantitate HR activity (assay vs universal mix, according to the manufacturer’s instructions). Data in panels B and D are mean ± SD of 3 independent experiments. *P < .05, ** P < .01, ***P < .0001, unpaired Student t test.