Figure 5
miR-124 regulates mitochondria activity via Vimentin. Primary motor neurons of control, miR-124 overexpression alone or transduced in addition with lentiviral vector for Doxycyclin-dependent expression of Vim (>80% transduction efficacy, transduction 24 hrs. before transfection of miRNA mimics), without or with the chemical inducer (Dox). (a) Vim mRNA and (b) VIM protein expression without or with Dox. Three experimental repeats, Averages \(\pm \) SEM, Student’s t-test. A full view of the Western blot is in Sup. Figure 6. (c) Confocal fluorescent micrographs of primary motor neurons, stained with MitoTracker (green) and TMRE (red), merged signal (yellow) and nuclei (blue, Hoechst). Scale bars, 20 μm. Bar graphs quantification of fluorescent signal intensity in (d) axons and (e) soma. Quantification of three random positions per compartment; 9 neurons per condition; Data from three replicates and the study performed in three independent experimental repeats. Averages ± SEM, ANOVA + Duncan’s new multiple range test (MRT) *P-value < 0.05, **p < 0.01, ***p < 0.001. (f) Snapshot from mitochondria live imaging along axons of primary motor neurons. Bar graphs quantification of (g) axonal mitochondria density/μm and (h) mean TMRE intensity in control axons (n = 48), miR-124 overexpression alone (n = 52), miR-124 and Vim without Dox (n = 34) or with Dox (n = 34). Averages \(\pm \) SEM, Student’s t-test, P-value *p < 0.05, **p < 0.01.
