Pten deficiency enhances megakaryopoiesis in vivo and differentially affects NOTCH-induced megakaryopoiesis from LSK versus CMP cells. (A) Flow cytometric analysis of myeloid progenitors within the Lineage−cKit+Sca1− population of PtenFlox/Flox-Mx1Cre− (Pten+/+) or PtenFlox/Flox-Mx1Cre+ (Pten−/−) animals 2 weeks after pIpC. A representative of 5 independent animals is shown for each group. (B) Whole BM cells from Pten+/+ and Pten−/− mice were plated in MegaCult for assessment of CFU-MK potential. Mean ± SEM of quadruplicate experiments is represented. (C) Immunohistochemical analysis for the VWF highlights megakaryocytes (black). (D) Bar graphs are a representation of the analysis performed in panel C. The mean ± SEM number of megakaryocytes per microscope field in 25 independent fields is shown. (E) Megakaryocytes from Pten+/+ and Pten−/− mice were analyzed for ploidy content with the use of propidium iodide. Left and middle panels: a representative analysis is shown. Percentages of polyploid cells (> 4n) are indicated. Right panel: Bar graphs are representation of 4 independent analyses. (F) LSK cells from Pten+/+ and Pten−/− mice were flow-sorted 2 weeks after pIpC treatment and were cultured on OP9-DL1 stroma in the presence or absence of 1μM GSI. After 6 days of cocultures, cells were analyzed by flow cytometry for the development of CD41+ cells within the CD45+ gate. (G) CMP cells from Pten+/+ and Pten−/− mice were flow-sorted 2 weeks after pIpC treatment and analyzed as in panel F. (H) LSK cells from Pten+/+ and Pten−/− were cultured on OP9-DL1 stroma for 4 days and analyzed for phospho-FOXO by flow cytometry. Top panel: a representative analysis gated on CD45+ hematopoietic cells is shown. Bottom panel: bar graphs indicate the mean ± SEM of 2 independent experiments. (I) CMP cells from Pten+/+ and Pten−/− were analyzed as in panel H.
