Expression of ezrin^Y145F slows down the cell cycle of HS2 leukemic proerythroblasts. (A) Cell-cycle analysis of 606HS2-neo, 606HS2-Ez^Y145F, and 606HS2-Ez^WT cells. Cells were seeded at 2 × 10⁵ cells/mL. After 24 hours of culture, cells were ethanol-fixed, and the DNA content was estimated after PI incorporation by flow cytometry. Percentage of G₀/G₁, S, and G₂/M phases calculated with ModFit software is indicated. Graphs (FlowJo representation) for each clone are representative of 3 independent experiments. Similar results were obtained after 48 hours. (B) Inhibition of Akt activation in 606HS2-Ez^WT, 606HS2-Ez^Y145F, and 606HS2-neo cells cultured in the presence or absence of LY294002 (10 μM) for 24 hours. Cells were fixed with cytofix cytoperm buffer (BD Biosciences, Le Pont de Claix, France) and labeled with anti–phospho-Akt. The primary antibody was revealed with the anti-rabbit Alexa-488–conjugated antibody (Jackson ImmunoResearch, Montluçon, France) before flow cytometry analysis. Control corresponds to cells incubated with the secondary Alexa-488–conjugated antibody. Graphs are representative of 2 independent experiments. (C) Activation of Erk1/2 in 606HS2-Ez^WT, 606HS2-Ez^Y145F, and 606HS2-neo cells. Total cell extracts were subjected to immunoblotting with antibodies indicated on the right of the blots. Western blots are representative from 3 independent experiments.