MCL-1 expression and its regulation in AML LSCs. (A) Real-time PCR analysis of MCL-1, BCL-2, and BCL-xl expression within fractions of CD34⁺ cells. The LSC-enriched CD34⁺CD38⁻ fraction in FLT3-WT AML (cases 1, 3-5, 8-10, 16, 18, and 19) expressed Mcl-1 at a level > 6-fold higher than that of normal HSCs, whereas LSCs in FLT3-ITD AML (cases 20, 22-24, 27, and 30) expressed even ∼ 2-fold higher levels of MCL-1 compared with FLT3-WT LSCs. In contrast, BCL-2 or BCL-xL expression was not significantly changed. (B) Cytoplasmic staining of MCL-1 in CD34⁺CD38⁻ AML fractions with FLT3-ITD and FLT3-WT. A high amount of MCL-1 protein is detectable in FLT3-ITD LSCs. Representative FLT3-ITD and FLT3-WT LSCs are shown. (C) Changes in the expression level of MCL-1 in normal HSCs, LSCs of FLT3-WT AML, and of FLT3-ITD AML after incubation with FL or with SCF. Each bar represents an n-fold difference in the amount of MCL-1 expression prior to stimulation of normal HSCs. Results are mean ± SD (*P < .05).