MDS/MPN phenotype in lyn^−/−;PLC-β3^−/− mice. (A) Flow cytometric analysis of nucleated cells in BM, spleen (SPL), and peripheral blood leukocytes (PBL) at the age of 3 months. (B) Graphic summary of granulocytes and monocytes (CD11b⁺ cells) in these organs of 2- to 4-month-old lyn^−/−;PLC-β3^−/− mice (n = 12 per cohort). Data are mean ± SD, *P < .05. (C) Spleens at the age of 4 months. (D) Mature granulocytes and monocytes in blood smear at the age of 4 months. (E) Hematoxylin and eosin staining of monolobated megakaryocytes in the BM of lyn^−/−;PLC-β3^−/− mice, compared with multilobated ones in wt mice. (F) Graphic summary of megakaryocytes with the indicated numbers of nuclear lobes in BM at the age of 4-6 months (n = 3).

Phenotypic similarities between lyn^−/−;PLC-β3^−/− and me^v/me^v mice. (A) Flow cytometric analysis of granulocytes and monocytes in BM and peripheral blood from 6-week-old me^v/me^v mice. (B) Immunofluorescent staining for F4/80 and Ym-1 in lung tissues. Bar indicates 50 μm. (C) BM cells were subjected to flow cytometric analysis for HSCs/progenitors. The absolute number was calculated based on the percentage of KSL cells in BM. (D) BM cells were stimulated with IL-3 and then phospho-Stat5 levels in KSL cells were analyzed by flow cytometry (n = 4). (E) Sorted CD34⁻KSL cells (50 cells/well) were cultured in 96-well plates in the presence of IL-3 and SCF. Data are mean ± SD, *P < .05.

Tyr⁵⁶⁴ phosphorylation is critical for catalytic activity and biologic functions of SHP-1. (A) CD34⁻KSL cells from me^v/me^v mice were retrovirally transduced with empty vector or various SHP-1 mutants. Sorted GFP⁺ cells were cultured in the presence of IL-3 and SCF for 4 weeks and then measured for SHP-1 phosphatase activity. Data are mean ± SD, *P < .05 versus vector control. SHP-1 levels shown are taken from Figure 5B. (B) Stat5 phosphorylation in the cells from panel A was analyzed by flow cytometry. Stat5 levels in the cell panel were assessed by immunoblotting. (C) CD34⁻KSL cells from me^v/me^v mice were transduced with a bicistronic retroviral vector encoding wt SHP-1, DYF SHP-1, Y536F SHP-1, Y564F SHP-1, or empty vector, together with GFP. Transduced cells were sorted and cultured in the presence of IL-3 and SCF. (D) Transduced me^v/me^v CD34⁻KSL cells were adoptively transferred (without sorting) to lethally irradiated C57BL/6-Ly5.1 mice. Donor-derived (Ly5.2⁺) CD11b⁺ cells in peripheral blood were analyzed by flow cytometry.

Premature death and lung inflammation in lyn^−/−;PLC-β3^−/− mice. (A) Lyn phosphorylation at Tyr³⁹⁶ was measured in BM cells by immunoblotting. (B) Survival analysis of lyn^−/−;PLC-β3^−/− mice. The numbers of mice used: wt, 12; PLC-β3^−/−, 36; lyn^−/−, 16; lyn^−/−;PLC-β3^−/−, 32. (C) Immunofluorescent staining for F4/80 and Ym-1 in frozen sections of lung tissues. DAPI (4′,6-diamidino-2-phenylindole) is nuclear staining. Bar indicates 50 μm. (D) Cells in bronchoalveolar lavage fluid (BALF) were analyzed for CD11b and F4/80 expression by flow cytometry. The total cell and CD11b⁺F4/80⁻ cell numbers were calculated (n = 4-7). Data are mean ± SD, *P < .05, **P < .01.

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.

Lyn and PLC-β3 regulate SHP-1 phosphorylation at Tyr⁵³⁶ and Tyr⁵⁶⁴. (A) Scheme of a panel of SHP-1 retroviral constructs. (B) CD34⁻KSL cells from me^v/me^v mice were retrovirally transduced with empty vector or SHP-1 mutants. GFP⁺ cells were sorted and cultured in the presence of IL-3 and SCF for 4 weeks and were analyzed by immunoblotting. Right 2 lanes indicate results with nontransduced cells. (C) BM cells were analyzed by flow cytometry for phospho-Tyr⁵³⁶ and phospho-Tyr⁵⁶⁴. MFI of SHP-1 phosphorylation in KSL cells was shown (n = 6). Data are mean ± SD, *P < .05 versus wt. (D) BMMCs were analyzed by immunoblotting. (E) wt CD34⁻KSL cells were retrovirally transduced with PLC-β3 CT. GFP⁺ cells were sorted and cultured in the presence of IL-3 and SCF for 4 weeks. Interactions among PLC-β3, Lyn, and SHP-1 were examined by coimmunoprecipitation followed by immunoblotting. (F) Left, PLC-β3^−/− CD34⁻KSL cells were retrovirally transduced with PLC-β3 CT. GFP⁺ cells were sorted and cultured in the presence of IL-3 and SCF for 4 weeks. The interaction among PLC-β3, Lyn, and SHP-1 was examined by coimmunoprecipitation followed by immunoblotting. Right, splenocytes were used for coimmunoprecipitation and immunoblotting. (G) GST-SHP-1 (0.5 μg) was incubated with various amounts of recombinant Lyn protein and analyzed by immunoblotting. (H) me^v/me^v BMMCs expressing the indicated SHP-1 proteins were immunoprecipitated with anti–SHP-1 and followed by immunoblotting.

Reduced SHP-1 phosphatase activity is responsible for MDS/MPN development in lyn^−/−;PLC-β3^−/− mice. (A) CD34⁻KSL cells were cultured in the presence of IL-3 and SCF for 4 weeks and were analyzed for SHP-1 phosphatase activity. Data are mean ± SD, *P < .05 versus vector control. Similar expression levels of SHP-1 among the cells can be seen in Figure 5D. (B) CD34⁻KSL cells from lyn^−/−;PLC-β3^−/− mice were retrovirally transduced with empty vector or various SHP-1 mutants. Sorted GFP⁺ cells were cultured in the presence of IL-3 and SCF for 4 weeks and then measured for SHP-1 phosphatase activity. (C) Stat5 phosphorylation in the cells from panel B was measured by flow cytometry. Expression of SHP-1 (C) and Stat5 (D) was assessed by immunoblotting. (D) CD34⁻KSL cells from lyn^−/−;PLC-β3^−/− mice were transduced with a bicistronic retroviral vector encoding wt SHP-1, DYE SHP-1, or empty vector, together with GFP. Transduced GFP⁺ cells were sorted and cultured in the presence of IL-3 and SCF. (E) Transduced lyn^−/−;PLC-β3^−/−CD34⁻KSL cells were adoptively transferred (without sorting) to lethally irradiated C57BL/6-Ly5.1 mice. Donor-derived (Ly5.2⁺) CD11b⁺ cells in peripheral blood were analyzed by flow cytometry. **P < .01.