PDK1 is a critical regulator of MK ploidy and proplatelet formation in vitro and in vivo. (A) Representative ploidy histogram (n = 6) of Pdk1^fl/fl MKs (black trace) and Pdk1^−/− MKs is shown. (B) DNA content in Pdk1^fl/fl and Pdk1^−/− CD41⁺ BM-derived MKs cultured for 5 days (left). Arithmetic means ± SEM (n = 6, 2-way ANOVA with Bonferroni correction for multiple comparisons) are shown. Percentage of Pdk1^fl/fl and Pdk1^−/− CD41⁺ BM-derived MKs with a DNA content greater (right bars) and smaller (left bars) than 8N (right). Arithmetic means ± SEM (n = 6, 2-way ANOVA with Bonferroni correction for multiple comparisons) are shown. (C) Quantification of the percentage of proplatelet-forming BM-derived MKs from Pdk1^fl/fl and Pdk1^−/− mice in vitro. Representative immunostainings of proplatelet-forming MKs from Pdk1^fl/fl (upper) and Pdk1^−/− mice (lower) are shown (left). Red, F-actin (Phalloidin-Atto647N); green, α-tubulin (anti–α-tubulin-Alexa488), cyan, (DAPI) nuclei. Scale bar equals 40 μm. Pdk1^fl/fl and Pdk1^−/− MKs extending proplatelets were counted 16, 24, and 48 hours after bovine serum albumin gradient and expressed as percentage of total MKs per field of view (right). Arithmetic means ± SD (n = 3, unpaired 2-tailed Student t test). (D) 2P-IVM revealing reduced proplatelet formation in Pdk1^−/− MKs in vivo. Platelets and MKs were stained with anti-GPIX antibody derivatives (green), and the vessel lumen was labeled using fluorescein isothiocyanate (FITC)–bovine serum albumin and anti-CD105 antibodies (red) (left). Representative median projections of 30-µm z-stacks from the BM of Pdk1^fl/fl and Pdk1^−/− MKs are depicted. Proplatelet-forming MKs (white arrows indicating proplatelets) were counted, and the ratio per mouse was assessed (arithmetic means ± SEM, n = 5 to 6 ice, 4 to 10 z-stacks per mouse, unpaired 2-sided Welch test; right). Scale bar equals 50 µm. *P < .05 and **P < .01 indicate statistically significant differences.