Figure 1.

Mitochondrial bioenergetic function reduction, ROS levels increase, mitochondrial membrane potential impairment and abnormal intermediate filament network in sacsin KO cells. Measurement of OCR (A), basal respiration, ATP production and proton leak (B) in WT and KO cells using the Agilent Seahorse XF Cell Mito Stress Test. The assay was performed under basal conditions and after addition of olygomycin (2 µM), carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP) (1.5 µM) and rotenone plus antimycin A (1 µM). Comparison between WT and sacsin KO cells showed impaired mitochondrial function (OCR = oxygen consumption rate; oligo = oligomycin; Rot = rotenone; aA = antimycin A). (C) Fluorimetric detection of intracellular ROS in WT and sacsin KO cells in basal condition (only addition of 2′,7′–dichlorofluorescin diacetate (DCFDA), 25 µM) and after tert-butyl hydroperoxide (TBHP) treatment (150 µM) using DCFDA assay kit showed a significant increase in intracellular ROS levels in KO cells after oxidative stress induction. Hoechst 33342 was used to normalize cell number. (D) Cells were loaded with the fluorescent cationic probe tetramethylrhodamine methyl ester (TMRM). TMRM, whose fluorescence intensity was measured using the Spectramax iD3 microplate reader, showed a significantly reduced Δψm in KO cells. Δψm was normalized by DAPI fluorescence, as a function of number of cells. (RFU = relative fluorescence units; Δψm = mitochondrial membrane potential). *p < 0.05; **p < 0.01; and ***p < 0.001. (E) Representative images of vimentin network (in red) in WT and sacsin KO cells showed a collapsed intermediate filament network in cells lacking sacsin. DAPI (in blue) was used as nuclear stain. Scale bar = 10 µm. (F) Western blotting showed undetectable sacsin levels in KO cell line. GAPDH was used as a loading control. Full-length blots are presented in the Supplementary Information 1.