Increased proliferation, decreased apoptosis and MPN-causing ability of lyn^−/−;PLC-β3^−/− HSCs are due to increased Stat5 activity. (A-B) BM cells were subjected to flow cytometric analysis of HSCs, myeloid, and erythroid progenitors. The absolute number was calculated based on the percentage of KSL cells and other progenitors in total BM cells. Results shown are representative of at least 3 measurements (2-4 months old, n = 12). (C) Sorted CD34⁻KSL cells (50 cells/well) were cultured in 96-well plates in the presence of IL-3 and SCF. (D) BM cells were stained for Lineage cocktail, c-Kit, Sca-1, and Annexin V, and subjected to flow cytometric analysis. Annexin V⁺ cells were counted in KSL populations (n = 4). (E) Sorted KSL cells were stained for either Pyronin-Y/Hoechst 33 342 (left) or propidium iodide (PI; right), and analyzed by flow cytometry. (F) BM cells were stimulated with IL-3, and phospho-Stat5 levels were analyzed by flow cytometry. Mean fluorescence intensity (MFI) of Stat5 phosphorylation in KSL cells is presented (n = 6). (G) Sorted CD34⁻KSL cells were cultured in methylcellulose in the presence or absence of IL-3. Ten days later, the number of colonies (including CFU-G, CFU-M, and CFU-GM) was counted. (H) Sorted CD34⁻KSL cells from BM of lyn^−/−;PLC-β3^−/− mice were transduced with a bicistronic retroviral vector encoding DN Stat5 or empty vector, together with GFP. Transduced (GFP⁺) cells were sorted into 96-well plates (50 cells/well) in the presence of IL-3 and SCF. (I) Sorted CD34⁻KSL cells from lyn^−/−;PLC-β3^−/− mice were transduced with DN Stat5 or empty vector and then adoptively transferred (without sorting) to lethally irradiated C57BL/6-Ly5.1 mice. Four months later, CD11b⁺ donor-derived (Ly5.2⁺) cells in peripheral blood were analyzed by flow cytometry. Data are mean ± SD, *P < .05, **P < .01.