SRC is essential for FLT3-ITD–mediated STAT5 activation and proliferation. (A) Cells (32D) stably expressing FLT3-ITD or FLT3-TKD were retrovirally infected with an SRC, LCK, or control siRNA–encoding vector and selected with puromycin. To ensure the down-regulation of SRC or LCK, immunoblotting of whole-cell lysates was performed using an SRC or LCK Ab, respectively. STAT5 activation was investigated in the same manner using pSTAT5 and STAT5 Abs. FLT3 and ACTIN immunoblotting was conducted to ensure equal loading. (B) Proliferation of 32D cells stably expressing FLT3-ITD or FLT3-TKD in combination with SRC or control siRNA was analyzed after 48 hours using the MTT assay. IL-3 stimulation of the FLT3-ITD + SRC siRNA-expressing cells for 48 hours served as a control. Data were correlated to cells expressing the control siRNA and represent values ± SD of triplicates. One representative of at least 3 independent experiments is shown. Statistical analysis was conducted. ***P < .001; **P < .01; *P < .05; and n.s. (no significance), P > .05. (C) Proliferation of FLT3-ITD⁺ 32D cells transduced with an SRC, LCK, or control siRNA. Analysis was performed as in panel B. (D) Kaplan-Meier plot detailing survival times of C3H mice injected with 32D FLT3-ITD cells stably expressing either control siRNA or SRC siRNA (n = 10). The mice were closely monitored and killed when moribund.