Enforced Pim-1 expression renders BaF3/ITD cells more resistant to cytotoxicity induced by CEP-701 while dominant-negative Pim-1 expression inhibits colony growth. (A) Triplicate samples of 50 000 cells were incubated with increasing concentration of CEP-701 for 24 hours, and cytotoxicity was assessed by MTT assay. The graph displays the MTT results for each cell line normalized to the untreated controls. Error bars represent standard deviation from 3 independent experiments. (B) Pim-1/NT81 inhibits colony formation by FLT3/ITD. BaF3/ITD cells were electroporated with 15 μg of the expression vectors for the N-terminal deletion form of Pim-1 (Pim-1/NT81) or control vector (pLXSN). One day after electroporation, 100, 500, and 2500 cells were seeded on triplicate dishes containing fetal calf serum (FCS), IMDM, 1% methylcellulose, and G418. The colonies were grown at 37°C in 5% CO₂ and counted on day 7. The mean colony numbers are counted from triplicate dishes of 100, 500, and 2500 plated cells. The graph shows the relative mean colony number difference and standard deviation of Pim-1/NT81 transfectants compared with control vector transfectants from each group from 3 independent experiments. The inset shows one each of the 10-mm culture dishes from control vector and Pim-1/NT81 electroporations. (C) Cells were incubated with increasing concentrations of CEP-701 for 24 hours and assayed for viability by annexin V and 7-AAD binding by fluorescence activated cell sorting (FACS) analysis. The graph displays the percentage of cell death induced in each cell line by the treatment normalized to the untreated controls. Error bars represent standard deviation from 3 independent experiments.