(A-a): SN4741 represents dopaminergic neuronal cells and DJ-1(−/−) represents dopaminergic neuronal cells homozygous for a DJ-1 gene deletion. To compare the native state of the respiratory complexes in these cell lines, isolated mitochondria were solubilized by n-dodecyl-β-d-maltoside (DDM) and resolved by using blue native-polyacrylamide gel electrophoresis (BN-PAGE). After transferring to a nitrocellulose membrane, the blot was incubated with monoclonal antibodies against five mitochondrial complex subunits using MS601 antibody cocktail (MitoSciences, Eugene, OR). The molecular weight of mitochondrial complex I was about 1000 kDa. Normalization of isolated mitochondria was achieved based on HSP60 protein expression level. The result shown is representative of three independent experiments. (A-b): Protein expression levels were analyzed quantitatively by densitometry. Error bars represent the mean ± SD. Significant difference compared to SN4741 cells: ***, p<0.001. (B): Isolated mitochondria solubilized by digitonin were analyzed by BN-PAGE for supercomplex detection. Supercomplex was detected by using MS601 monoclonal antibody cocktail as described above. I+III₂+IV: supercomplex formed of complex I, dimeric complex III, and complex IV. I+III₂: supercomplex formed of complex I and dimeric complex III. III₂+IV: supercomplex formed of dimeric complex III and complex IV. III₂: supercomplex formed of dimeric complex III (C-a): 2D gel analysis. BN-PAGE was performed with mitochondria isolated from SN4741 cells or DJ-1 null cells to obtain the first dimension of resolved native complexes. By using BN-PAGE gels strips, further separation in the second dimension was performed by SDS-PAGE. In DJ- null cells, in the complex I lane of the 2D gel, the uppermost dot is missing; compare the circles marked by asterisks in the two panels. (C-b,c): Quantification of complex I ‘dot’ levels. Two dots present in SN4741 cells were compared to the dots of DJ-1 null cells in the same position. The upper dot levels in each cell line were normalized to the lowest dot in the complex I lane of the respective cell lines and the value obtained for the DJ-1 null cells was compared to that of the SN4741 cells; the results are derived from three independent experiments. (D-a, b): The spot on the gel was analyzed by mass spectroscopy and determined to be the complex I subunit NDUFS1.

(A-a, b): The O₂ consumption rate (OCR) was measured in SN4741 cells and DJ-1 null cells in at least three independent experiments by using an XF analyzer. For validation of the measured O₂ consumption rate, we used the 2 µg/ml oligomycin, 5 µM CCCP, and 1 µM rotenone sequentially. Each time point represents the mean (±SD), and compensated OCR data without background levels are shown in (b) with bar graphs. ***, p<0.001 (B): Mitochondrial membrane potential was investigated by rhodamine 123 staining and quantified by FACS analysis. ***, p<0.001 (C): The cell pellet was solubilized by digitonin and the mitochondrial ATP production rate was measured by a luminometer. To confirm that the calculated luminescence values represented ATP content, oligomycin was used to inhibit ATP production. *, p<0.05; **, p<0.01.

(A): To identify mitochondrial morphologic changes, SN4741 cells and DJ-1 null cells were stained with MitoTracker red and co-stained with antibodies to COX4, which is a subunit of complex IV. The nucleus was identified by DAPI staining. The cells were sequentially observed by confocal microscopy at a magnification of ×400. (B-a): Cells were fixed, cryosectioned, and observed by transmission electron microscopy (TEM). DJ-1 null cells exhibit smaller mitochondria, which is apparent at a higher magnification (inset, ×50,000). The original magnification was ×15,000. (B-b): Mitochondrial area was analyzed by Image J software. A minimum of 20 TEM slides was evaluated to characterize mitochondrial dynamics. We divided the section equally between the largest area and the smallest area of mitochondria and measured the number of mitochondria in that section. Compared to SN4741 cells, most of the DJ-1 null cells had smaller mitochondria. (C-a, b): Mitochondrial mass was evaluated by MitoTracker green staining and quantified by FACS analysis. DJ-1 null cells showed a left shift in the curve, indicating reduced mitochondrial mass. Significant differences were consistently observed in three independent experiments. ***, p<0.001 (D): Rotenone (10 nM), an inhibitor of complex I, did not induce changes in mitochondrial complex native protein as assessed by BN-PAGE analysis. HSP60 was used as a loading control. *** p<0.001.

(A): Forty-eight hours after transfection of AdDJ-1, overexpression of DJ-1 protein was confirmed by western blotting. (B-a, b, c): Mitochondrial structure was evaluated by TEM and quantified by mitochondrial area and number. (C-a): Overexpression of AdDJ-1 rescued mitochondrial complex I structure, as revealed by BN-PAGE. (C-b): Statistical analysis of protein expression levels showed a significant recovery by AdDJ-1 compared to transfection with mock virus. (D): The ADP/ATP ratio was measured by fluorometry using an ApoSensor kit. *, p<0.05; **, p<0.01; *** p<0.001.