Defective Autophagy, Mitochondrial Clearance and Lipophagy in Niemann-Pick Type B Lymphocytes
Fig 3
Autophagic Vacuole (AV) detection and endocytic compartment evaluation.
(A) Acidic vesicular organelle (AVOs) detection by LysoTracker Green (LTG) in flow cytometry. (i) Statistical histogram of MFI variation of LTG in tWT and tNP cells for each experimental condition. Mean values were converted to arbitrary units (A.U.) setting control of wild-type cells as 100. Each value is expressed as a relative mean ± SD (Results from n ≥ 3 independent experiments). The difference between cell lines was determined to be significant by two-way ANOVA (***P < 0.001). Two-way ANOVA with Bonferroni post test revealed a P value < 0.01 (++P < 0.01) between tWT and tNP starved cells (ii) Flow cytometry histogram overlay showing lysotracker green MFI values of tWT and tNP basal condition. (iii) Fold increase related to histogram in (i) starved and rapamycin-treated cells in both cell lines, versus respective control. (B) Autophagic vacuole detection by Monodansylcadaverine (MDC) in fluorescence microscopy and flow cytometry. (i) Microscopy images showing monodansylcadaverine fluorescence from control and starved tWT and tNP cells. White arrows indicate cells rich in AV. Bars: 10μm. (ii) Statistical histogram depicting MFI variation of monodansylcadaverine in tWT and tNP cells for control, starved and rapamycin-treated cells. Mean values were converted to arbitrary units (A.U.) setting control of wild-type cells as 100. Each value is expressed as a relative mean ± SD (Results from n ≥ 3 independent experiments). Two-way ANOVA with Bonferroni post test revealed a P value < 0.05 (+P < 0.05) between tWT and tNP in basal condition and rapamycin treated and a P value < 0.01 (++P < 0.01) between tWT and tNP after starvation treatment (iii) Monodansylcadaverine flow cytometric histograms are overlaid to show the comparison among MFI values of basal condition, starved and rapamycin-treated samples in tWT (upper panel) and tNP (lower panel) lymphocytes. (C) TEM autophagic vacuole detection in control condition of tWT (a, b) and tNP (c-g) cells. Black arrows indicate AVs. Bars: 2 μm for a, b, c, d, f, g; 0.5 μm for e. (D) TEM autophagic vacuole detection in normal (a, e) and NP-B B lymphocytes (b, c, d, f), after nutrient deprivation (e, f) and rapamycin administration (a-d). Black arrows indicate AVs. Black arrowhead indicates a lipid droplet inside AV. Mvb: multivesicular bodies. Bars: 1 μm for a, f; 2 μm for b, c, d, e. (E) Double- and single- membrane autophagic vacuoles from tWT and tNP lymphocytes were counted in control, starvation and rapamycin conditions. Each value is expressed as an absolute number (70 cells counted/experiment). Two-way ANOVA with Bonferroni post test revealed a P value < 0.01 between tWT and tNP basal condition and a P value < 0.05 between tWT and tNP rapamycin treated.
