(a) LN229 cells transfected with control siRNA (Ctrl) or SNPH-directed siRNA were stained with antibodies against Tom20, β-tubulin and DAPI and analysed by confocal microscopy. Top, 3D rendering of mitochondria localization by fluorescence microscopy. Bottom, 3D drawings represent the cells above, and show mitochondrial repositioning to the cortical cytoskeleton. Scale bar, 10 μm. (b,c) Cells were labelled with MitoTracker (b) or Tom20 (c) and analysed for mitochondrial recruitment to the cortical cytoskeleton. Symbols correspond to individual cells. Data are represented as mean±s.e.m. **P=0.003; ***P<0.0001 by Student's t test. (d) PC3 cells transduced with the indicated shRNA were transfected with vector or shRNA-insensitive SNPH cDNA and analysed for mitochondrial trafficking to the cortical cytoskeleton. Symbols correspond to individual cells. ***P<0.0001 by one-way ANOVA and Bonferroni's post test. (e,f) Cells transfected with control or SNPH-directed siRNA and expressing mitochondria-targeted RFP were imaged by time lapse microscopy, and individual mitochondria were tracked through the stack to calculate trafficking parameters (e), and the speed of individual mitochondria per condition (f). Data are represented as mean±s.e.m. (n indicated on the individual bars). ***P<0.0001 by Student's t test. (g,h) The displacement of mitochondria relative to the initial location was calculated in 2D plots (g) and the distance travelled by individual mitochondria was quantified (h). Each tracing on g represents individual mitochondria and are colour coded according to Euclidean distance displacement. Each dot on h correspond to individual mitochondria (n indicated in the individual panels). ***P<0.0001 by Student's t test.