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Fig. 2.
Co-localization of DJ-1 with mitochondrial complex I. (A) SH-SY5Y GS1101 were stained with Mito Tracker Red and then with an anti-DJ-1 antibody. Nuclei were stained with DAPI. Cells were then reacted with FITC- or rhodamine-conjugated secondary antibody and images were visualized under a fluorescence microscope as described in Materials and methods. (B) NIH3T3 cells were heated at 95 °C for 15 min and stained with anti-DJ-1 and anti-NDFFS3 antibodies. Cells were then reacted with FITC- or rhodamine-conjugated secondary antibody and images were visualized under a fluorescence microscope as described in Materials and methods.
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Reduction of complex I activity in DJ-1-knockdown cells
Since DJ-1 was associated with complex I, it is possible that DJ-1 affects complex I activity. Expression levels of DJ-1 and subunits of mitochondrial complexes in NIH3T3 cells and their knockdown cells D2 were first examined by Western blotting. About 80% reduced expression of DJ-1 was observed in D2 cells compared to the expression of DJ-1 in NIH3T3 cells (Fig. 3B). Difference of expression levels of three subunits of complex I, NDUFA9, NDUFS3, and ND6, and a subunit of complex V, ATP5A1, between D2 and NIH3T3 cells were much smaller than that of DJ-1. Activities of complex I in these cells were then measured, and it was found that the activity in D2 cells was only about 30% of that in NIH3T3 cells (Fig. 3A).