Inhibition of MEK/ERK pathway increased Bim-EL levels and reduced cell viability of Nras/CM leukemic cells. (A) Western blot analysis of Bim-EL, phospho-Akt, Akt, phospho-Erk1/2, and Erk1/2 levels in Nras/CM leukemic cells treated for 5 hours with dimethyl sulfoxide (Con), MEK inhibitor PD325901 or PI3K inhibitor GDC-0980 (left); quantification of Bim-EL protein levels normalized to Erk1/2 is shown for 3 independent Nras/CM clones (right). A vertical line has been inserted to indicate a repositioned gel lane. (B) Bcl2l11 transcript levels after the same 5-hour treatment were analyzed by quantitative RT-PCR and values were normalized to Actb; this experiment was performed in 3 independent Nras/CM clones, each in triplicate (average value of each clone relative to control is shown in the figure). (C) Quantification of apoptosis (Annexin V⁺/7AAD^–) levels in c-kit⁺-gated Nras/CM leukemic cells treated for 24 hours; this experiment was performed in 3 independent Nras/CM clones, each in triplicate (average value of each clone relative to control is shown in the figure). (D-E) Dose-response curve of Lin^– wild-type BM (open circle), CM leukemic cells (open square), or Nras/CM leukemic cells (open triangle) to (D) MEK inhibitor (PD325901) or (E) PI3K inhibitor (GDC-0980) for 48 hours. Cell viability was measured by CellTiter-Glo assays. Nonlinear regression curve fit was generated by inhibitor (log) vs normalized response-variable slope analysis. The mean of 3 independent clones from each group is shown. *P < .05, **P < .01.