ITD mutations suppress PU.1 expression and function.

(A) Real time RT-PCR analysis of PU.1 mRNA from 32D/Flt3-WT (○) and 32D/Flt3-ITD (●) stimulated with IL-3 or FL as indicated for 36 hours or more. The expression level is shown in the same way as in Figure 6. (B) Western blot analysis of Pu.1 protein from 32D/Flt3-WT and 32D/Flt3-ITD stimulated with cytokines for 48 hours. (C) Analysis of Pu.1-transactivating activity on a Pu.1-responsive promoter construct, 3 × WT-MHC-luc. 32D/Flt3-WT and 32D/Flt3-ITD were starved from IL-3 for 2 hours and transfected with 3 × WT-MHC-Luc and pRLnull. After stimulation with the indicated cytokines for 16 hours, cells were lysed, and the luciferase activity of the cell lysates was determined. The relative luciferase activity was normalized to renilla luciferase activity (activity of cotransfected pRLnull vector). med indicates medium, no cytokines.