Figure 5.

Aurka loss increased the number of CD41⁺Mpl⁺ megakaryocytes and megakaryocyte differentiation. (A) Approximately 3 × 10⁴ Lin^-c-Kit⁺Sca-1^- cells were analyzed. The distribution of MEPs and megakaryocytes in the BM of mice with different genotypes was assessed by flow cytometry. The data shown are representative of one of two independent experiments. (B) The dot graph shows the number of MEPs and megakaryocytes in BMs removed from the Aurka^f/f mice or the Aurka^f/f;Cd19^Cre/+ mice. The data shown are representative of one of two independent experiments (n = 7 mice/group) with similar results. Significance was calculated using an unpaired Student's t-test. *, P < 0.05. (C, left) H&E and Immunofluorescence staining for CD41⁺Mpl⁺ megakaryocytes, CD19⁺ B cells, or CD138⁺ plasma cells in the BM sections from the Aurka^f/f or Aurka^f/f;Cd19^Cre/+ mice. (C, right) The dot graph shows the number of CD41⁺Mpl⁺ megakaryocytes in the BM sections from the Aurka^f/f mice or the Aurka^f/f;Cd19^Cre/+ mice. The data shown is representative of one of two independent experiments (n = 3 mice/group, 5 slides/mouse) with similar results. P values were obtained using a 2-sided, unpaired Student's t-test. **, P < 0.01. (D) Dot graph shows the number of CD19⁺ B cells or CD138⁺ plasma cells within the 25 mm distance with respect to megakaryocytes in the BM sections from the Aurka^f/f mice or the Aurka^f/f;Cd19^Cre/+ mice. The data shown is representative of one of two independent experiments (n = 3 mice/group, 5 slides/mouse) with similar results. Significance was calculated using an unpaired Student's t-test. n.s., not significant. (E) The ploidy of megakaryocytes was studied after staining with anti-CD41, anti-Mpl antibodies and Hoechst labeling. The histograms show one of three independent experiments (n = 5 mice/group) with similar results. (F) The ultrastructure of megakaryocytes was visualized by TEM. DM, demarcation membrane; N, nucleus; A, α-granules. The data shows one of three independent experiments (n = 3 mice/group) with similar results. (G) Lin^-c-Kit⁺ cells from the BM of either the Aurka^f/f mice or the Aurka^f/f;Cd19^Cre/+ mice were cultured in the presence of 200 ng/mL mTPO for 4 days. (Left) Representative staining of megakaryocytes with anti-CD41 (green) and anti-vWF (red) antibodies was shown. (Right) Proplatelet forming megakaryocytes were counted. The data shows one of two independent experiments (n = 3 - 5 mice/group) with similar results. Significance was calculated using an unpaired Student's t-test. **, P < 0.01. (H) Lin^-c-Kit⁺ cells from the BM of the Aurka^f/f mice were cultured with CD19⁺ B cells sorted from either the Aurka^f/f mice or the Aurka^f/f;Cd19^Cre/+ mice in the presence of 200 ng/mL mTPO together with murine IL-17 for 4 days. Proplatelet-forming megakaryocytes were counted. The dot graph shows one of two independent experiments with similar results. Significance was calculated using an unpaired Student's t-test. * P < 0.05.