Increased FOXO1 promotes a preleukemic state in human CD34⁺HSPCs . (A) Weekly cell count of liquid culture of CD34⁺ HSPCs transduced with FOXO1 WT, DB, or control (MIG) retroviral vectors. FOXO1 expression was confirmed by immunoblot. One representative experiment of 3 replicates is shown. (B) Flow cytometry analysis of the myeloid differentiation marker CD11b on cells from a week-4 culture. (C) Wright-Giemsa staining of week-4 cultured cells. The images were obtained using Motic BA310 microscope with ×40 objective. Bar represents 20 μm. (D and E) CFU assay with transduced cells. Results represent mean ± SD of colony counts of first round (D; *P < .05, **P < .001) and second and third rounds (E; *P < .01, **P < .005); n = 3. (F) Transduced cells were transplanted into immunodeficient mice. GFP⁺ percentage of human engrafted cells in bone marrow was examined 12 weeks later and normalized to those before the transplantation. *P < .05; **P < 1 × 10⁻⁵. One representative experiment of 2 replicates is shown. All P values were calculated by unpaired 2-tailed Student t test.