Figure 4. CDC25A is an important determinant of FLT3-ITD leukemic cells proliferation.

a. MV4-11 and MOLM-14 FLT3-ITD positive cells, KG1, HL-60 and TF-1 FLT3 wild type cells, and K562 FLT3 negative cells were cultured in the presence of the CDC25 inhibitor IRC-083864 (200 nM). Cells were harvested each day and counted after trypan blue staining. The graph represents three independent experiments. b. Cell death was estimated by trypan blue staining in cells (MOLM-14; MV4-11; MOLM-14 TKD) treated with IRC-083864 200 nM for 48 hours. c. MOLM-14 and MV4-11 cells were transfected with CDC25A siRNA for 24 hours, and cells were counted after trypan blue coloration. The efficiency of CDC25A siRNA was estimated by western blot analysis (inserts). ns: non specific. d. Primary cells from patients were cultured in semi-solid medium to estimate their clonogenic potential as described in the Methods section, in the presence or the absence of IRC-083864 (100 and 200 nM). 8 FLT3-ITD positive (upper panel) and 8 FLT3-wild type (lower panel) AML primary samples were used for these experiments. Leukemic colonies were scored under an inverted microscope at day 7. e. MOLM-14, and MOLM-14 TKD cells, generated as described in Supplementary material and methods, were grown in the presence of AC-220 1 nM (upper panel) or IRC-083864 200 nM (lower panel). Cells were harvested each day and counted after trypan blue staining. The graphs represent three independent experiments.