Fig. 5.

Mitochondrial dynamics and oxidative metabolism were altered in the SPG4 patient derived fibroblasts. a, b Western blot analysis showed a significant difference in spastin isoforms (M87 and M87△Ex4) expression levels between the SPG4 patient and WT. c ICH of mitochondria maker (ATP synthase, red) and DAPI (blue) in fibroblasts from the WT (upper panel) and the SPG4 patient (lower panel). Insets in the images are enlarged (original magnification, ×8.0) to the right. Scale bar, 10 μm. d Quantification of mitochondria distribution measured from the center of the cells (n = 3, > 10 cells per experiment). The data showed that the number of mitochondria were significantly reduced along the branch in the SPG4 patient derived fibroblasts. e Quantification of cells with different mitochondrial morphology (tubular, intermediate and fragmented patterns) to total cell (n = 3, > 30 cells per experiment). f, g Western blot analysis showed a significant difference in VDAC1 protein expression levels between the SPG4 patient and WT. h JC-1 staining was used to measure MMP (Δψm) in fibroblasts from the WT (upper panel) and the SPG4 patient (lower panel). Boxed areas are enlarged in the insets. Scale bar, 50 μm. i Statistical analysis of h (n = 3). j Intracellular ROS visualization using DCFH-DA staining in WT (upper panel) and SPG4 (lower panel). Scale bar, 50 μm. k Statistical analysis of (j) (n = 3). l ATP bioluminometers analysis revealed a significant decrease of ATP content in the SPG4 patient. Error bars indicate SEM. *, P < 0.05