MEK1 knockdown by shRNA blocks ERK1/2 activation and sensitizes MM cells to γH2A.X expression and lethality induced by Chk1 inhibitors. (A,B) U266 cells were stably transfected with constructs encoding MEK1 shRNA or a scrambled sequence as a control, and exhibited down-regulation of MEK1 expression by WB (top panels). Cells were then incubated with UCN-01 or Chk1i for 24 hours, after which WB analysis was performed to detect expression of phospho-ERK1/2 and γH2A.X, as well as PARP degradation. Blots were subsequently stripped and reprobed for expression of α-tubulin to ensure equivalent loading and transfer of protein. Additional 2 studies yielded equivalent results. CF indicates cleavage fragment. (C,D) Alternatively, after 24-hour and 48-hour treatment, flow cytometry was performed to monitor apoptosis (annexin V–FITC staining). Results represent the means plus or minus SD for 3 separate experiments performed in triplicate. *P < .05; **P < .01; NS indicates no significance (P > .05).