Defective Autophagy, Mitochondrial Clearance and Lipophagy in Niemann-Pick Type B Lymphocytes
Fig 7
Detection of microvesicles and lipid particles in the extracellular environment and analysis of lysosomal exocytosis.
(A) Statistical histogram showing lipid particle counting by NR-FL2 in the extracellular environment for each experimental condition. Each value is expressed as an absolute number ± SD (Results from n ≥ 3 independent experiments); *P < 0.05 vs respective control. Two-way ANOVA with Bonferroni post test revealed a P value < 0.05 (+P < 0.05) between tWT and tNP starved cells. (B) Statistical histogram showing CD63+ events counted in the extracellular environment for each experimental condition. Each value is expressed as an absolute number ± SD (Results from n ≥ 3 independent experiments). (C) Extracellular microparticle/microvesicle detection by FC. (a) Dot plot FSC vs SSC in logarithmic visualization. P1: viable cells; P2: Dako CytoCount counting beads 5.2 μm diameter; P3: beads 1 μm diameter; P4: beads 0.5 μm diameter; (b) Density plot FSC vs FL1 of the negative sample; (c) Density plot FSC vs FL1-CD63 of starved and (d) rapamycin-treated tWT cells. (e) Density plot FSC vs FL1-CD63 of starved and (f) rapamycin-treated tNP cells. (D) Statistical histogram of surface Lamp-1/CD107a detection on fresh cells: MFI values shown are for each experimental condition and from both cell lines. Each value is expressed as a mean ± SD (Results from n ≥ 3 independent experiments); *P < 0.05 and **P < 0.01 vs respective control. Two-way ANOVA with Bonferroni post test revealed a P value < 0.05 (+P < 0.05) between tWT and tNP basal condition and a P value < 0.001 (+++P < 0.001) between tWT and tNP starved cells.
