Figure 3.

Strong discrepancy between in vitro cultured proplatelet formation and in vivo native proplatelet formation in Tubb1-/- mice. (A) Upper panel, explant bone marrow culture experiment. Representative phase contrast images and quantification of the percent of megakaryocytes (MK) presenting with cultured proplatelets (cPPT) after 6 hours of culture showing total absence of Tubb1-/- MK with protrusions (0%) while 55% of wild-type (WT) MK extended cPPT (mean ± standard error of the mean [SEM], n=3 independent experiments). Lower panel, MK forming PPT after 4 days of Lin– progenitor culture. Representative bright field images and quantification of the percent of MK presenting with cPPT (mean ± SEM, n=4 independent experiments). Inset, rare Tubb1-/- MK forming abnormal cPPT without shaft extensions. (B) Estimation of the nPPT formation capacity in situ by calculating the ratio of PPT fragments over the total number of MK observed in 30-mm thick marrow sections. Bars are mean ± SEM of three to four independent marrow sections from two mice, representing 374 and 687 MK for WT and Tubb1-/-, respectively. (C) Two z-projection images of in vivo Tubb1-/- native proplatelets (nPPT). Dotted lines delimit the sinusoids. Representative of at least 12 nPPT from five mice. (D) nPPT elongation speed. Data are mean ± SEM of 16 nPPT pooled from eight WT mice and 12 nPPT pooled from four Tubb1-/- mice. (E) nPPT length. Data are mean ± SEM of 25 nPPT pooled from 14 WT mice and 16 nPPT pooled from five Tubb1-/- mice. (F) nPPT width. Data are mean ± SEM of 28 nPPT pooled from 19 WT mice and 16 nPPTs pooled from five Tubb1-/- mice. Bar graphs represent the mean ± SEM; all data analyzed using Mann-Whitney test. ns: not significant.