Figure 7. Increased reactive oxygen species (ROS) production in DJ-1−/− MEFs.

(A, B) Confocal microscopy analysis of ROS concentration. (A) Representative confocal live cell images of DJ-1−/− and +/+ MEFs after incubation with Mitotracker Green (200 nM) and Amplex Red (2.5 µM), DHEt (2.5 µM) or MitoSOX Red (2.5 µM). Scale bar: 10 µm. (B) The bar graph shows the quantification and the increase of Amplex Red, DHEt or MitoSOX Red fluorescence in DJ-1−/− cells compared to control cells. The number shown in the panel indicates the number of cells quantified per genotype in the study. (C) Kinetics analysis of ROS production. The time course of the fluorescence changes in DJ-1−/− and +/+ MEFs labeled with Amplex Red (upper), DHEt (middle), or Mitotracker CM-H₂XROS (lower) is shown. The bar graph at the bottom shows quantitative analysis of fluorescence changes, indicating significant increases of fluorescence signals of Amplex Red, DHEt and Mitotracker CM-H₂XROS in DJ-1−/− MEFs. The number shown in the panel indicates the number of embryos used to derive primary MEFs per genotype, and the data were obtained from four independent experiments. (D) Kinetics of H₂O₂ production in isolated mitochondria measured by following Amplex Red fluorescence over time showing an increase of its production in DJ-1−/− MEFs. The number shown in the panel indicates the number of embryos used to derive primary MEFs per genotype, and the data were obtained from three independent experiments. All data are expressed as the mean ± S.E. *p<0.05, ***p<0.001.