FIGURE 4.

Introduction of phosphomimetic Cytc into Cytc knock-out cells leads to controlled respiration, intermediate mitochondrial membrane potentials, and low ROS levels. A, Western blotting of Cytc double knock-out lung fibroblasts stably transfected with empty vector (EV) and WT, T28A, and T28E expression constructs. B, proliferation rates of stably transfected lines were determined by cell counts; means ± S.D. (error bars) are reported; *, p < 0.05. C, intact cell oxygen consumption rates (OCR) are decreased in both Cytc mutants. D, mitochondrial membrane potential as determined by JC-1 fluorescence was decreased in cells expressing T28E versus WT and T28A Cytc. E, basal mitochondrial reactive oxygen species generation as determined with MitoSOX is decreased in cells expressing T28E Cytc compared with WT. F, ATP levels determined with the bioluminescent method were decreased in cells expressing T28E Cytc compared with WT. G, live cell counts after treatment with H₂O₂ indicate that T28E-expressing cells are better protected at intermediate H₂O₂ levels compared with WT, whereas T28A-expressing cells show decreased survival at all concentrations compared with WT. Error bars, S.D.