Figure 4
Vimentin regulates neuron morphology and mitochondria function (a) Venn diagram of predicted miR-124 targets (TargetScan) and transcripts that were experimentally repressed >2 fold by miR-124 overexpression in primary motor neurons, relative to control conditions. (b) qPCR analysis of the three mRNAs that obeyed both criterions: Vim, Ptbp1 and Mdk, in primary motor neurons transfected with miR-124 oligos (N = 3), relative to controls (N = 3). (c) miR-124 recognition element at Vim 3′UTR is conserved in vertebrate species. Knockdown of Vim by shRNA lentiviruses53 downregulated (d) Vim mRNA and (e) VIM protein expression, relative to non-targeting shRNA controls. A full view of the Western blot is in Sup. Figure 6. (f) High content image analysis of cell numbers, mean axonal outgrowth per cell and mean number of branches per cell. 100 Tuj1+ neurons quantified per field, 8 fields/well, 6 replicates per treatment. Data collected from >24,000 Tuj1+ neurons per treatment. (g) Confocal fluorescent micrographs of primary motor neurons, 72 hrs. post transduction with scrambled-shRNA or Vim-shRNA, stained with TMRE (red) and nuclei (blue, Hoechst). Transduction efficacy >80%. Scale bars, 20 μm. Bar graphs quantification of fluorescent signal intensity in (h) axons and (i) soma. Quantification of 3 random positions per compartment; 9 neurons per condition. Data from three replicates and the study performed in three independent experimental repeats. All graphs present averages \(\pm \) SEM, Student’s t-test. *P-value < 0.05, **p < 0.01, ***p < 0.001.
