Absence of FOXO factors increases megakaryocytic potential of LSK cells and CMPs in vivo. (A) Immunohistochemical analysis of BM sections was performed against the VWF to highlight the megakaryocytes in FoxO1/3/4^Flox/Flox-Mx1Cre⁻ (FoxO^+/+) versus FoxO1/3/4^Flox/Flox-Mx1Cre⁺ (FoxO^−/−) animals. Bar graphs represent the mean ± SEM number of megakaryocytes per microscope field in 25 independent fields. (B) Representative images of analysis performed in panel A. Megakaryocytes are in black. (C) Whole BM cells from FoxO^+/+ or FoxO^−/− animals were plated in MegaCult for assessment of CFU-MK potential. Mean ± SEM of quadruplicate experiments is represented. (D) Quantitative expression analysis of Gata-1 in flow-sorted LSK cells and CMPs from FoxO^−/− or FoxO^+/+ animals. All signals were normalized to Gapdh and are shown relative to FoxO^+/+ RNA. Bar graphs represent the mean ± SEM of triplicate experiments. (E) Flow cytometric analysis of myeloid progenitors within the Lineage⁻cKit⁺Sca1⁻ population of FoxO^+/+ or FoxO^−/− animals 3 weeks after pIpC. A representative analysis is shown. (F) LSK cells from FoxO^+/+ or FoxO^−/− mice were flow-sorted 3 weeks after pIpC treatment and cultured on OP9-GFP or OP9-DL1 stroma in the presence or absence of 1μM GSI. After 6 days of coculture, cells were analyzed by flow cytometry for the development of CD41⁺ cells within the CD45⁺ gate. Bar graphs indicate the mean ± SEM of 4 independent experiments. (G) CMP cells from FoxO^+/+ or FoxO^−/− mice were purified and analyzed as in panel F. Bar graphs indicate the mean ± SEM of 4 independent experiments.