Cytotoxic effect is not exclusively dependent on inhibition of FLT3 autophosphorylation. (A) MTT assay dose-response curves for Sample 3 (circles) and Sample 13 (squares) incubated with AC220 (open symbols) and lestaurtinib (filled symbols). Error bars represent standard deviations. (B) Blasts from Sample 3 (top blots) and Sample 13 (bottom blots) were incubated for 1 hour in culture medium with the indicated concentrations of lestaurtinib (left) and AC220 (right). The blasts were then analyzed for phospho-FLT3 and phospho-STAT5 as described in “FLT3 phosphorylation.” (C) Blasts from Sample 3 were thawed and resuspended in culture medium. An aliquot was removed (Day 0), fixed, and stained with anti-CD33, anti-CD3, anti-CD19, and annexin V for flow cytometric analysis, and then the remaining blasts were divided into 3 cultures: DMSO control, lestaurtinib 50 nM, and AC220 50 nM. After 72 hours of culture, the blasts were fixed, stained, and analyzed for comparison with the day 0 blasts. Shown are the forward (x-axis) and side (y-axis) scatter plots. Gate A represents dead cells (annexin V⁺); gate B, viable blasts (annexin V⁻, CD33⁺); gate C, lymphocytes (CD3⁺ or CD19⁺).