Functional analysis of MCL-1 in FLT3-ITD AML. (A) MCL-1 expression in cell lines with FLT3-WT (NB4, HL-60, U937) and FLT3-ITD (MV4-11 and MOLM-13). High levels of MCL-1 mRNA and protein were seen in MV4-11 cells by real-time PCR analysis (left panel), the intracellular MCL-1 staining, and Western blot analysis (right panels). (B) The inhibition of MCL-1 protein (top) and cell survival (bottom) in MV4-11 after PKC-412 treatment. The inhibitory effect of PKC-412 reached plateau at a concentration of 0.5μM. Apoptotic cell death was evaluated by annexin/PI staining. Vertical lines have been inserted to indicate a repositioned gel lane. (C) The enforced expression of MCL-1 prevented FLT3-ITD cell lines from apoptosis in the presence of PKC-412. MCL-1 transcript and protein expression were significantly up-regulated 48 hours after transduction of MCL-1 into MV4-11 cells. PKC-412–induced apoptotic cell death is significantly inhibited in MV4-11 cells (right panel) (*P < .05). (D) Inhibition of MCL-1 by shRNA resulted in an apoptosis of FLT3-ITD cell lines. Forty-eight hours after transduction of MCL-1–shRNA–EGFP into the MV4-11 cells, MCL-1 transcript (left) as well as MCL-1 protein expression on FACS and Western blot analysis (middle) was significantly down-regulated. In parallel, the number of apoptotic cells increased by ∼ 2-fold compared with control (right). Results are shown as mean ± SD (*P < .05). RE indicates relative expression levels.