Figure 4.

Oxidative stress induces mitochondrial caspase-2-like activity. NIH 3T3 cells were transfected with a (a) combination of mCFP and mYFP, or (b) mCGY, or (c) mC2Y and analyzed for FRET. (a) Cartoon showing the mechanistic basis of the mitochondrial-targeted constructs. Close proximity of CFP and YFP will result in FRET that is lost when mC2Y is cleaved by mitochondrial caspase-2 under conditions of oxidative stress. mCGY cannot be cleaved by caspase-2 and demonstrates FRET activity constitutively. (b) Representative FRET images of cells transfected with either mCGY or mC2Y in the presence of 100 μM tert-butyl hydroperoxide (t-BuOOH). (c) Analysis of FRET efficiency after treatment with 100 μM t-BuOOH for 1 h. (d) Analysis of FRET efficiency after treatment with 0.5 μM staurosporine (STS) for 5 h. Data are representative of three independent experiments in which 5–10 fields were analyzed per cell and 5–10 cells were quantified per experiment.