Pim-1 decreases in response to FLT3 inhibition in EOL-1, MV4-11, TF1/ITD, BaF3/ITD, and 32D/ITD cells. Cell lines were incubated for increasing times or with increasing concentrations of CEP-701 or CEP-5214. RNA was then harvested and used for Pim-1 expression analysis by QPCR. GAPDH or ribosomal S16 gene expression were used as internal controls to normalize the level of Pim-1 expression. Inhibition of Pim-1 expression by 50 nM CEP-701 with increasing time of treatment in (A) the constitutively activated FLT3-expressing human leukemia-derived cell lines, EOL-1, MV4-11, TF1/ITD; and (B) the constitutively activated FLT3/ITD-transformed murine cell lines, BaF3/ITD and 32D/ITD. (C) Changes in Pim-1 expression in EOL-1 cells after treatment and release from 50 nM CEP-5214 inhibition. EOL-1/C indicates control; EOL-1/4h, 4-hour inhibition; EOL-1/4R, 4-hour release after 4 hours of inhibition; EOL-1/8h, 8-hour inhibition; EOL-1/8R, 8-hour release after 8 hours of inhibition. (D) Pim-1 response in BaF3/ITD cells to increasing concentrations of CEP-701 treatment for 2 hours.