Stromal protective effect on cell death by FLT3 inhibitors. (A) MV4-11 cells were incubated for 72 hours with the indicated concentrations of G-749, AC220, and PKC412 in CM supplemented with 35% HS-5–derived medium or normal culture medium (NM). Cell viabilities were then determined by test inhibitors and plotted for comparison between NM and CM. (B) AML blasts (patient 12, supplementary Table 4) were cultured with HS-5 monolayer for 48 hours with each test inhibitor at 250 and 1000 nM. All cells were stained with annexin V (x-axis) and CD45 (y-axis) and then gated for CD45 positivity. The percentages of live and apoptotic AML blasts were indicated in the upper left and upper right, respectively. (C) Molm-14 cells were incubated for 6 hours in CM with the indicated concentrations of G-749 or AC220. The phosphorylation levels of FLT3, STAT5, AKT, and ERK1/2 were detected by immunoblotting. Noticeable differential response is that G-749 potently inhibited p-ERK1/2, but AC220 did not.