Vps34 is critical for MK migration and granule biogenesis. (A) TEM of MKs from native bone marrow sections. Images are representative of 5 mice of each genotype. Scale bar represents 5 µm (upper images) and 1 µm (lower images). The α-granule number and mean area were quantified on 70-µm2 field of TEM images by using ImageJ software (mean ± SEM; n = 5 mice of each genotype; **P < .01; ***P < .001 vs WT according to 2-tailed Student t test). (B) WT or Vps34-deficient (Vps34) MKs were exposed to a SDF1α gradient within the Dunn chamber. Migration paths over 6 hours of 5 representative MKs from 8 independent experiments in each graph were traced. The intersection of the x-axis and y-axis was taken to be the starting point of each cell path, whereas the source of the SDF1α was at the top. The accumulated distance and directionality from WT MKs in the presence of dimethyl sulfoxide (WT) or 1 μM VPS34-IN1 or from Vps34-deficient (Vps34) MKs were analyzed by using the ImageJ software manual tracking plug-in (mean ± SEM; n = 30-50 MKs from 8 independent experiments; **P < .01 vs WT according to 1-way ANOVA). (C) Representative confocal images of immunostained native bone marrow. MKs and platelets are labeled by vWF staining (green). FABP4/A-FABP staining (red) labels sinusoid vessels. Nucleus staining was done by 4′,6-diamidino-2-phenylindole (blue). Scale bars represent 50 μm. The graph represents the percentage of platelets inside and outside the sinusoids (mean ± SEM; n = 20 images from 4 mice of each genotype, ***P < .001 vs WT according to 2-tailed Student t test).
