FIGURE 5.

Mitochondrial function and GPx-1 overexpression. A, to determine global protein disulfide bond formation, GPx-1-overexpressing and control cells were cultured on chamber slides, fixed with methanol, processed to block free thiols with iodoacetamide, and incubated in tris(2-carboxyethyl) phosphine to reduce disulfides. The resulting free thiols were tagged with 5-iodoacetamidofluorescein according to the protocol of Yang et al. (23). The cells were imaged on a fluorescence microscope. CCCP, a mitochondrial uncoupler, was added to some wells to demonstrate the role of mitochondrial activity in the formation of disulfides. B, mitochondrial potential was determined in cells cultured in 96-well plates in the presence of 2 μm JC-1, a dye that accumulates in the mitochondria proportionate to mitochondrial membrane potential. The ratio of red fluorescence (mitochondrial JC-1) to green fluorescence (cytoplasmic JC-1) was used as a surrogate for mitochondrial potential. GPx-1 OE have a significantly lower JC-1 ratio (p < 0.01, by t test). C, adenovirus treatment of cells to enhance GPx-1 expression resulted in a dose-dependent decrease in JC-1 ratio (p < 0.005) compared with control. The values are plotted compared with no adenovirus treatments, which was set to 100%. JC-1 ratio for BGal adenovirus was not significantly different from no virus control.