Fig. 5.

Opa1^TetraCys MEFs are protected from H₂O₂ induced cell death.

(A) MEFs of the indicated genotype were treated as indicated, collected, stained with annexin V/PI and analyzed by cytofluorimetry. Boxes represent SEM with mean values, whiskers 10th-90th percentiles. Values of the individual independent experiments are plotted as dots. *, p < 0.05 in a two sample t-test between the indicated groups.

(B) Pseudocolor-coded images of TMRM fluorescence in MEFs of the indicated genotypes treated with 1 mM H₂O₂. The pseudocolor scale is indicated. Top images represent the initial frame of the real-time sequence, while bottom ones were acquired at time = 40 min. Bar, 40μm.

(C) Quantitative analysis of TMRM fluorescence changes over mitochondrial regions in experiments as in B. Where indicated, 1 mM H₂O₂ and 2 μM FCCP were added. Data represents mean ± SEM of 4 independent experiments.

(D) Representative confocal images of cytochrome c subcellular distribution. MEFs of the indicated genotype were transfected with mtRFP (red), treated where indicated with 1 mM H₂O₂ for 30 min, fixed and immunostained for cytochrome c (green). Bar, 15 μm

(E) Localization index of cytochrome c in MEFs of the indicated genotype treated where indicated with 1 mM H₂O₂ for 30 min. Plots represent mean ± SEM of 9–13 independent experiments performed as in D. Open dots indicate values of individual experiments. *, p < 0.05 in a one way ANOVA with Tukey's mean comparison between the indicated conditions.

(F) Mitochondria (0.5 mg/mL) isolated from MEFs of indicated genotype were incubated with 100 μM H₂O₂ for the indicated time. Upon crosslinking with 10 mM EDC, equal amounts (15 μg) of mitochondrial proteins were separated by SDS-PAGE and immunoblotted using the anti Opa1 antibody. Asterisks indicate the Opa1 oligomer. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)