Loss of Sphk2 results in extramedullary thrombopoiesis but does not change MK development, serum Tpo levels, or platelet life span. (A) Quantification of CFU-MK numbers in BM cells. Representative of 6 independent experiments performed in triplicate. (B) Representative immunostaining of MKs in mouse femoral BM and spleen. The samples were examined using a Leica microscope equipped with ×20 objective lens (numerical aperture [NA] = 0.5) or ×10 objective lens (NA = 0.3) and a commercial charge-coupled device (CCD) camera. Images were acquired by Axiovision software. MKs were detected by the MK-specific marker CD41 (green); 4,6-diamidino-2-phenylindole (blue); scale bar represents 10 µm. (C) Quantification of MK numbers per ×20 high-power field in femoral BM sections (n = 3 for each group). (D) Quantification of MK numbers per ×10 high-power field in spleen (n = 3 for each group). (E) The ratio of spleen weight to whole body weight (left) and the representative picture of extracted spleens from WT or Sphk2^−/− mice (right) (n = 5 for each group; 16-20 weeks old). (F) The percentage of MKs with DNA ploidy (2N, 4N, and ≥8N) in WT and Sphk2^−/− mice (n = 3 mice for each group). (G) Serum Tpo levels (n = 3 for each group). (H) Platelet life span of WT and Sphk2^−/− mice (n = 3 for each group). All error bars represent SEM.