Fissional/fragmental changes of mitochondrial structure in DJ-1 null cells.

<p><b>(A):</b> 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>(B-a):</b> 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-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. <b>(C-a, b):</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 <b>(D):</b> 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.