Analysis and comparison of FDC-P1 cells expressing different KIT receptor mutants. (A) High vector expression levels, as determined by FACS analysis of YFP expression, were observed in FDC-P1 cells expressing EC or TM mutant, as compared with other KIT mutants. Shown is the mean fluorescence of 2 independent experiments. (B) IL-3–independent FDC-P1 cells expressing EC and TM KIT receptor mutants express high KIT protein levels, as compared with cells expressing other KIT mutants. Protein from total cell lysates was analyzed by Western blot analysis with anti-KIT antibody. Tubulin levels served as a loading control. (C) Proliferation response of FDC-P1 cells expressing wild-type or mutant KIT to imatinib. 3H-thymidine incorporation in controls (dimethyl sulfoxide [DMSO] only) was set at 100% for each culture. Shown is one representative experiment of 3, each performed in triplicate. (D) Apoptosis detection after imatinib treatment of parental FDC-P1 cells with or without IL-3 (FD + IL-3 or FD - IL-3) or factor-independent FDC-P1 cells expressing mutant KIT. Surface expression of annexin V was measured 12 hours after drug treatment. IL-3 withdrawal from control FDC-P1 cells showed significant slower kinetics of apoptosis. Shown is a representative experiment of 3 independent experiments. (E) Phosphorylation status of the KIT receptor and downstream signaling targets was determined by Western blot analysis using a phospho-specific antibody after imatinib treatment (3 hours). Due to the different levels of KIT protein in the various cell cultures, exposure times for the KIT blot were varied accordingly. For controls, protein extracts from FDC-P1 cells maintained in IL-3 or FDC-P1 transduced with wild-type hKIT and maintained in stem cell factor (SCF) were analyzed. (F) Proliferation assays were performed in the presence of the indicated inhibitor for 48 hours to determine the importance of the different signaling pathways for proliferation.