Figure 5. Increased opening of mitochondrial permeability transition pore in DJ-1−/− cells.

(A, B) Confocal microscopy analysis. (A) Representative confocal microscopic images of DJ-1−/− and +/+ MEFs after incubation with calcein-AM (1 µM, green) and Mitotracker Red (150 nM) in the presence or absence of Co²⁺ (1 mM), which quenches calcein fluorescence (green) outside of mitochondria. Mitotracker Red confirms the localization of calcein fluorescence in mitochondria. Insets indicate higher power views of the boxed area in the panel. The calcein fluorescence in mitochondria is lower in DJ-1−/− cells in the presence of Co²⁺. In the absence of Co²⁺, calcein fluorescent signals are very intense and are present in the entire cell, and there are no genotypic differences. Scale bar: 10 µm. (B) The bar graph shows quantification of calcein fluorescence in DJ-1−/− and +/+ cells in the presence or absence of Co²⁺. The number shown in the panel indicates the number of cells quantified per genotype in the study. (C, D) FACS analysis. (C) Representative flow cytometric dot plots show the intensity of calcein signal in DJ-1−/− and +/+ MEFs following incubation with calcein-AM (1 µM) in the presence or absence of Co²⁺ (1 mM). (D) The bar graph of calcein signal measured by FACS analysis shows reduced calcein signal in DJ-1−/− MEFs in the presence of Co²⁺. The number shown in the panel indicates the number of embryos used to derive primary MEFs per genotype, and the data were obtained from five independent experiments. All data are expressed as mean ± SEM. *p<0.05, ***p<0.001.