MLL-AF4 conveys a selective proliferation coupled to a survival advantage to CD34⁺ HSPCs. (A) In vitro cell growth, measured as cumulative population doublings, of purified GFP- versus MLL-AF4–expressing CD34⁺ HSPCs (n = 2). (Inset) RT-PCR confirming specific expression of the MLL-AF4 transcript only in the purified MLL-AF4⁺ CD34⁺ HSPCs. (B) Representative BrdU versus PI flow cytometry identifying the apoptotic (Sub-2n) cell fraction and the cells at different stages of the cell cycle: G₀/G₁ phase, S-phase, and G₂/M phase (n = 3). (C) Apoptosis and cell cycle distribution in GFP⁺ (transduced) and GFP⁻ (nontransduced) cell fractions sorted from EV-transduced CD34⁺ HSPCs (left panel) and MLL-AF4–transduced CD34⁺ HSPCs (right panel) (n = 3). (D) Representative flow cytometry panels displaying the apoptosis/cell death of EV- and MLL-AF4-transduced CD34⁺ HSPCs analyzed by 7-AAD and annexin-V costaining. (E) FACS mean fluorescence intensity (MFI) expression levels of the antiapoptotic proteins Bcl-2 and Bcl-xL in GFP⁺ cell fractions sorted from EV-transduced and MLL-AF4–transduced CD34⁺ HSPCs relative to GFP⁻ cell fractions.