Figure 3

FLT3 ITD signaling drives DUSP6 expression. MV4-11 cells endogenously expressing FLT3 ITD were subjected to different treatments. (A) Cells were transiently transfected with FLT3-specific siRNA using “Smart pool” siRNA and the nucleofection technology. After 48 h, DUSP6 protein levels were assessed by immunoblotting. Left panel: example experiment. Right panel: quantification of 2 independent experiments (*p < 0.05 by t-test). (B) MV4-11 cells were treated with the FLT3 inhibitor cpd.102 (1 μM) in complete medium for the indicated time periods. DUSP6 levels were assessed by immunoblotting. The experiment is representative of 2 with consistent results. (C) Cells were treated with the FLT3 inhibitors cpd.102 (1 μM), or AG1295 (20 μM), as indicated, in complete medium for different time periods. After treatment, cells were harvested, RNA was prepared, and DUSP6 expression was assessed by RT-qPCR relative to ACTB mRNA. Values are means + SD of duplicate determinations and are representative of 3 experiments with consistent results. (D) To assess reversibility of the inhibition of DUSP6 mRNA expression, cells were treated for 4 hours with AG1295 or solvent as in (C). Aliquots of the cells were directly subjected to analysis of DUSP6 mRNA expression (as indicated). The AG1295-treated cells were washed, and either cultivated without inhibitor (washout) or with freshly added inhibitor (as indicated) for another 4 h before analysis of DUSP6 mRNA expression. Values are means + SD of 4 independent determinations performed in triplicate. Statistic significance was tested by Students’ test (**p < 0.01, ***p < 0.001). (E) Cell treatments were performed with the MEK inhibitor UO126 (10 μM), and analysis carried out as in (C).