Growth and transforming capacity of 32D cell lines expressing ER-targeted FLT3. (A) 32D cell lines expressing the indicated FLT3 variants or parental 32D cells were seeded in 96-well plates (2.5 × 10⁴ cells/well), and cell growth in the absence or presence of FL (10 ng/mL) or IL-3 (2.5 ng/mL) was measured after 2 days using the MTT method. Values were normalized to growth in the presence of IL-3, which was set to 1.0 (means ± SD, n = 8). (B) The indicated versions of 32D cell pools were subjected to colony formation assays in methylcellulose in the absence or presence of FL or IL-3. Representative sections were photographed after 5 to 6 days of culture using equipment described in “Proliferation and colony-forming assays.” (C) Kaplan-Meier plot of survival of C3H/HeJ mice receiving parental 32D cells or 32D cells expressing the different FLT3 versions as indicated. Each group contained 8 to 10 mice. The percentage of surviving mice (y-axis) is plotted with respect to time in days (x-axis). Circles represent mice killed without a myeloproliferative disease or which served as controls at various time points, respectively. Statistical significance was tested using Gehan-Breslow statistics for the survival curves; post hoc comparisons were made with the Holm-Sidak method for all pairwise multiple comparisons. The reduction of survival in case of application of FLT3 ITD or FLT3 ITD R3–expressing cells was significant (P < .05) compared with all other groups. There was no statistical difference in survival between parental cells and any of the FLT3 D835Y–harboring cells, nor among the latter.