Pim-2 is an ITD target gene involved in ITD-mediated transformation.

(A-B) Flt3-ITD induces Pim-2 mRNA and protein selectively. (A) mRNA was obtained from 32Dcl3/flt3-ITD (●) and 32Dcl3/Flt3-WT (○) cells, which were stimulated with FL or IL-3 for the time indicated. The amount of Pim-2 mRNA was analyzed in each sample by real time RT-PCR. The ratio of expression compared with flt3-WT after normalization to GAPDH levels is shown. (B) Total lysates of 32D/flt3-ITD and 32D/flt3-WT stimulated with or without cytokines for 24 hours were electrophoresed and blotted with anti–Pim-2 antibody. The time point “start” indicates the starting point of the starvation period. The concentrations of FL and IL-3 were 50 ng/mL and 1 ng/mL, respectively. (C) Pim-2^K120M inhibits colony formation by Flt3-ITD. 32D/Flt3-ITD was electroporated with 15 μg of the expression vectors for kinase-defective Pim-2^K120M or control vectors and 2 μg pcDNA3. One day after electroporation, cells were seeded on 1% methylcellulose. The colonies were counted on day 8. The mean colony numbers and standard deviation of 3 experiments is shown.