Figure 5

DUSP6 attenuates ERK1/2 activity but positively regulates proliferation of FLT3 ITD expressing 32D cells. 32D cells stably expressing FLT3 ITD were subjected to transduction with lentiviral particles encoding shRNA targeting Dusp6. Transduced cell pools were selected, and levels were assessed by RT-qPCR for Dusp6 mRNA (relative to Actb) (A). The effect of Dusp6 knockdown for the indicated shRNAs on ERK1/2 phosphorylation was assessed in serum-starved cells by immunoblotting (B). Upper panel: Representative experiment, lower panel: quantification of 2 experiments (means + SD). pERK1/2 signals were normalized to ERK1 signals. Cell pools transduced with Dusp6-targeting shRNA were subjected to proliferation assays in 0.5% FCS in absence of cytokines with the MTT method. (C) Representative experiment, values are means of 7 replicas ± SD. (D) Summary of 4 independent experiments. Values at 72 h obtained for the two different Dusp6 shRNAs were normalized to the amounts measured in the individual experiments for the control shRNA cell pool (100%) (means + SD, n = 4, Statistic significance was tested by Students’ test (*p < 0.05).