Effects of AML1-ETO–mutNHR3 and –W692A on cellular dysregulation. (A) Effects of AML1-ETO mutants on apoptosis in primary bone marrow cells. Murine bone marrow cells transduced with the indicated AML1-ETO constructs were harvested after 2 d of selection, stained with 7-AAD and Annexin V-APC, and analyzed by flow cytometry. Gating controls are shown at left. Data are representative of 3 independent experiments. (B) Averages and standard deviations of 3 independent experiments from (A). (C) Effects of AE constructs on cellular metabolic activity. Primary bone marrow cells were transduced and selected as in (A), seeded in duplicate at 10 000 cells/well in a 96-well plate, and assayed every 2 d by MTS assay. Data are representative of 3 independent experiments. (D) Immunoblot analysis of CDK4, cyclins A and D3, and p27 expression in K562 cells infected and selected as in supplemental Figure 4A. Data are representative of 4 independent experiments. (E) W692A increases leukemogenic potential of AML1-ETO. Kaplan-Meier survival curves of mice transplanted with hematopoietic cells expressing the indicated AML1-ETO construct. Data combined from 3 independent transplantations. (F) Presence of hematopoietic blast cells in tissues of mice transplanted with AML1-ETO-W692A expressing cells. Peripheral blood smear and cytocentrifugation of bone marrow and spleen cells were stained with Wright-Giemsa solutions.