Defective Autophagy, Mitochondrial Clearance and Lipophagy in Niemann-Pick Type B Lymphocytes
Fig 2
Evaluation of mitochondrial dysfunctional features by flow cytometry and transmission electron microscopy.
(A) Statistical histogram of MFI variation of NAO in tWT and tNP cells for each experimental condition. Each value is expressed as a mean ± SD (Results from n ≥ 3 independent experiments); *P < 0.05 vs respective control. The difference between cell lines was significant as shown by two-way ANOVA (***P < 0.001). Two-way ANOVA with Bonferroni post test revealed a P value < 0.05 (+P < 0.05) between tWT and tNP in basal condition. (B) Flow cytometry histogram overlay depicting NAO MFI values of tWT and tNP basal condition. (C) Statistical histogram of MFI variation of TMRE in tWT and tNP cells for control, starved and rapamycin-treated cells. Mean values were converted to arbitrary units (A.U.) setting control values from wild-type cells as 100. Each value is expressed as a relative mean ± SD (Results from n ≥ 3 independent experiments); *P < 0.05 vs tWT control. The difference between cell lines was significant as shown by two-way ANOVA (*P < 0.05). Two-way ANOVA with Bonferroni post test revealed a P value < 0.05 (+P < 0.05) between tWT and tNP in basal condition. (D) Electron microscopy analyses of normal (a) and NP-B B lymphocytes (b-g) in control condition (a, b), and after starvation (c), rapamycin (e), nocodazole (f, g) and wortmannin (d) administration. In Niemann-Pick cells, dysfunctional mitochondria are recognizable (arrows): altered morphology is characterized by dilated and distorted cristae (c, d, e) and by stress-damaged features (b, f, g), absent in non-pathological cells (a). Bars: 1 μm for a, b, c, d, f, g; 0.5 μm for e. (E) Mitochondria from non-pathological and pathological cells were counted and classified according to their morphology—mitochondria with standard cristae (i), mitochondria with unusual cristae (ii) and oxidative stress-damaged mitochondria (iii)–in control, starved and rapamycin-treated cells. Each value is expressed as a percentage ± SD (n = 3; 50 cells counted/experiment); *P < 0.05 vs respective control. Two-way ANOVA with Bonferroni post test revealed a P value < 0.01 and < 0.05 for standard and unusual cristae numbers respectively (++P < 0.01 for standard cristae and +P < 0.05 for unusual cristae) between tWT and tNP in basal condition. (F) ROS detection by CM-H2DCFDA in flow cytometry. (i) Statistical histogram of MFI expression of CM-H2DCFDA in tWT and tNP cells. Mean values were converted to arbitrary units (A.U.) setting control of wild-type cells as 100. 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 in basal condition and after nocodazole treatment; NS: not significant. (ii) CM-H2DCFDA flow cytometric histograms are overlaid to show the comparison between MFI values of tWT and tNP basal condition. (G) Statistical histogram of MFI expression of MitoSOX in tWT and tNP cells. Mean values were converted to arbitrary units (A.U.) setting as 100 control MFI values of wild-type cells. Each value is expressed as a 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 after nocodazole treatment, and a P value < 0.01 (++P < 0.01) between tWT and tNP after starvation and wortmannin treatments; NS: not significant.
