Figure 3

Increased opening of the mitochondrial permeability transition pore in PINK1 −/− cells. A. Representative confocal microscopic images of PINK1−/− and +/+ MEFs after incubation with calcein-AM (1 μM) and Mitotracker Deep Red (150 nM) in the presence or absence of Co²⁺ (1 mM), which quenches calcein fluorescence (green) outside of mitochondria. Mitotracker Deep Red allows visualization of calcein fluorescence in mitochondria. The bottom right inserts are the higher power views of the boxed areas in the same panel. The calcein fluorescence in mitochondria is lower in PINK1−/− 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. B. The bar graph shows quantification of calcein fluorescence in PINK1−/− and +/+ cells in the presence or absence of Co²⁺ using confocal images. The number shown in the panel indicates the number of cells used in the study. C. Representative flow cytometry dot plots show the intensity of calcein signals in PINK1−/− and +/+ MEFs following incubation with calcein-AM (1 μM) in the presence or absence of Co²⁺ (1 mM). D. The bar graph of calcein signals measured by FACS analysis shows reduced calcein signals in PINK1−/− MEFs in the presence of Co²⁺. The number shown in the panel indicates the number of independent experiments performed. E. The bar graph of calcein fluorescent signals in PINK1−/− and +/+ neurons shows reduced calcein signals in PINK1−/− neurons. The numbers shown indicate the number of neurons used (left) and the number of independent experiments performed (right) in the study. All data are expressed as mean ± SEM. Scale bar: 10 μm. * p < 0.05, ** p < 0.01.