![Megakaryopoiesis is severely hampered in the absence of α4A- and β1-tubulins. (A) Flow cytometry analysis of platelet survival by in vivo biotinylation in WT, KOA4A (A4A), KOB1 (B1), and DKO mice. Graph represents the proportion over time of circulating biotin-labelled platelets. Results are expressed in %. Each symbol represents the mean ±SEM. N = 4 for each strain. Statistical analysis was conducted by 1-way ANOVA followed by Tukey HSD post-hoc test (not significant [ns]: P > .05). (B-C) Flow cytometry analysis of (B) megakaryocyte/erythroid progenitor (MEP) (Lin− CD16/32− Sca-1− ckit+ CD150+ CD9dim) and (C) megakaryocyte progenitor (MKP) (Lin− CD16/32− Sca-1− ckit+ CD150+ CD9bright) populations in freshly isolated, lineage-depleted bone marrow cell suspensions from WT, KOA4A (A4A), KOB1 (B1), and DKO mice. Results are expressed as % of the Lin− CD16/32− Sca-1− ckit+ cell population. Bar graphs represent the mean ±SEM. Each data point corresponds to an individual experiment. N = 3. Statistical analysis was conducted by 1-way ANOVA followed by Tukey HSD post-hoc test. (D) Megakaryocytes were manually counted from transmission electron micrographs of bone marrow sections of WT, KOA4A (A4A), KOB1 (B1), and DKO mice. Results are expressed as number of megakaryocytes per unit of surface (16 000 μm2). Bar graph represents the mean ±SEM. Each data point corresponds to an individual mouse. Statistical analysis was conducted by 1-way ANOVA followed by Tukey HSD post-hoc test. (E) Megakaryocytes from WT, KOA4A (A4A), KOB1 (B1), and DKO mice were manually classified (stages I, II, and III) based on morphologic criteria, evaluated from transmission electron micrographs of bone marrow sections. Results are expressed as % of each class. Stacked bar graph represents the mean ±SEM. N = 3 with at least 30 megakaryocytes counted per preparation. Statistical analysis of the stage III megakaryocytes was conducted by 1-way ANOVA followed by Tukey HSD post-hoc test, with DKO as a reference. ∗∗∗P < .001. (F) Representative transmission electron micrographs of stage III megakaryocytes observed in bone marrow sections of WT, KOA4A (A4A), KOB1 (B1), and DKO mice. Bottom panels show close-up views of the demarcation membrane system. Scale bar, 5 μm (top panels) and 2 μm (bottom panels).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/140/21/10.1182_blood.2022016729/5/blood_bld-2022-016729-gr2.jpeg?Expires=1725013268&Signature=VlnIlB4i7GKY-mLAd5mdNBMTOsOtHAuscCsIJfUUsCpRg64madVNEaFlOZO4iV4vFAUawQuET-xoKsRuO4dYJoDsiERQQT64p4pAaz1V-SiGBJuCUlSw66cfLxTkDhS1xSYDR3eHblQ8COa-TsDz2lquIdpVuo7L2dHspAGx2EecZdjlJSj1JFVHorLwa03STW1Acrv8yQf90b942ixQ5VZs33GXbFZJ-3Q7~WuQY5Stgug2SG0HCNYmMzuQ6-SFKNxIkdNBDdrKL9PkxObG7zAkTufnm~VXWnfvPyp6znSOAXAkaoA0XdAa0ifE6FYxhMHLNwInBwS8Wqs~7QFlEA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Megakaryopoiesis is severely hampered in the absence of α4A- and β1-tubulins. (A) Flow cytometry analysis of platelet survival by in vivo biotinylation in WT, KOA4A (A4A), KOB1 (B1), and DKO mice. Graph represents the proportion over time of circulating biotin-labelled platelets. Results are expressed in %. Each symbol represents the mean ±SEM. N = 4 for each strain. Statistical analysis was conducted by 1-way ANOVA followed by Tukey HSD post-hoc test (not significant [ns]: P > .05). (B-C) Flow cytometry analysis of (B) megakaryocyte/erythroid progenitor (MEP) (Lin− CD16/32− Sca-1− ckit+ CD150+ CD9dim) and (C) megakaryocyte progenitor (MKP) (Lin− CD16/32− Sca-1− ckit+ CD150+ CD9bright) populations in freshly isolated, lineage-depleted bone marrow cell suspensions from WT, KOA4A (A4A), KOB1 (B1), and DKO mice. Results are expressed as % of the Lin− CD16/32− Sca-1− ckit+ cell population. Bar graphs represent the mean ±SEM. Each data point corresponds to an individual experiment. N = 3. Statistical analysis was conducted by 1-way ANOVA followed by Tukey HSD post-hoc test. (D) Megakaryocytes were manually counted from transmission electron micrographs of bone marrow sections of WT, KOA4A (A4A), KOB1 (B1), and DKO mice. Results are expressed as number of megakaryocytes per unit of surface (16 000 μm2). Bar graph represents the mean ±SEM. Each data point corresponds to an individual mouse. Statistical analysis was conducted by 1-way ANOVA followed by Tukey HSD post-hoc test. (E) Megakaryocytes from WT, KOA4A (A4A), KOB1 (B1), and DKO mice were manually classified (stages I, II, and III) based on morphologic criteria, evaluated from transmission electron micrographs of bone marrow sections. Results are expressed as % of each class. Stacked bar graph represents the mean ±SEM. N = 3 with at least 30 megakaryocytes counted per preparation. Statistical analysis of the stage III megakaryocytes was conducted by 1-way ANOVA followed by Tukey HSD post-hoc test, with DKO as a reference. ∗∗∗P < .001. (F) Representative transmission electron micrographs of stage III megakaryocytes observed in bone marrow sections of WT, KOA4A (A4A), KOB1 (B1), and DKO mice. Bottom panels show close-up views of the demarcation membrane system. Scale bar, 5 μm (top panels) and 2 μm (bottom panels).
