Defective intracellular trafficking and PI3P production in Vps34-deficient MKs. (A-C) MK uptake of transferrin-Alexa Fluor546 (A) or fibrinogen-Alexa Fluor488 (B-C) was observed by confocal (A) or superresolution structured illumination microscopy (B-C) at different incubation time points. (A-B) Graphs represent fluorescence intensity quantified on a MK z-stack by ImageJ or Imaris software. (C) Representative 3-dimensional surface rendering of z-stacks acquired after 960 minutes of fibrinogen uptake are shown. Scale bar represents 5 µm. Graphs represent fibrinogen-positive structure number and volume quantified over time on 3-dimensional images using Imaris software (mean ± SEM; n = 10-60 MKs from 3 mice of each genotype; *P < .05; ***P < .001 vs WT according to 2-way ANOVA). (D-E) Fixed MKs stained with anti-clathrin, anti-EEA1, anti-Rab11, or anti-LAMP1 antibodies followed by corresponding secondary Alexa Fluor488 antibodies were observed by confocal microscopy. Live MKs were stained with LysoTracker Deep Red, fixed, and observed by confocal microscopy. Representative images of a z-stack are shown. Scale bar represents 5 µm. Graphs represent the structure number and area analyzed on a z-stack with ImageJ software (mean ± SEM; n = 30-50 MKs from 3 mice of each genotype; *P < .05; ***P < .001 vs WT according to 2-tailed Student t test). (F) PI3P mass assay performed on MKs as described in “Methods” (mean ± SEM; n = 5; **P < .01 vs WT according to 1-sample Student t test). (G) MKs stained with anti-PI3P and secondary Alexa Fluor488 antibodies were observed by confocal microscopy, and fluorescence intensity was quantified by using ImageJ software (mean ± SEM; n = 40 MKs from 3 mice of each genotype; ***P < .001 vs WT according to 2-tailed Student t test).
