Trilineage reconstitution by AKT1^−/−AKT2^−/− HSCs in long-term and serial competitive transplantations. (A) The number of methylcellulose colonies generated from equal numbers of LTC-ICs. LTC-ICs were derived by culturing sorted LSK cells for 2 weeks on OP9 monolayers. Graph represents the mean ± SEM (n = 3) of the number of colonies as a percentage of the control (AKT1^+/+AKT2^+/−) for each experiment. (B) The percentage of AKT1^−/−AKT2^−/− (CD45.2⁺Ly5^B6+) cells in the peripheral blood myeloid (Mac-1⁺Gr-1⁺), B (B220⁺), and T (TCRβ⁺) lineages 4, 6, 12, and 22 weeks after reconstitution. Graphs represent the mean ± SEM (n = 3) for each time point. (C) The percentage of AKT1^−/−AKT2^−/− (CD45.2⁺Ly5^B6+) cells in the LSK population 16 weeks after the initial transplantation (primary) and after 2 additional 16-week serial transplantations (secondary and tertiary). The graph represents the mean ± SEM (n = 4) of the percentage of CD45.2⁺Ly5^B6+ cells in the LSK population. The AKT1^+/+AKT2^+/− controls are the same samples represented in Figure 1C. (D) The percentage of AKT1^−/−AKT2^−/− (CD45.2⁺Ly5^B6+) cells in the bone marrow LSK subset and the splenic myeloid (Mac1⁺Gr1⁺), B (B220⁺), and T (TCRβ⁺) subsets 16 weeks after the secondary transplantation in the mice from panel C (secondary time point). The graph represents the mean ± SEM (n = 4). The LSK data are the same as in panel C (secondary time point). (E) Splenic γδT-cell competitiveness in the absence of AKT1 and AKT2. γδT cells were identified by gating on the singlet, DAPI⁻, Thy1⁺, γδTCR⁺ population. LSK data are the same as the primary transplantation in panel C. The graph represents the mean ± SEM (n = 4).