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. Author manuscript; available in PMC: 2013 Oct 22.
Published in final edited form as: J Proteome Res. 2008 May 17;7(6):2204–2214. doi: 10.1021/pr070371f

Figure 2. Examination of Mitochondrial Function Under Stress Conditions. 2A.

Figure 2

Index of mitochondrial integrity was assessed after reversible injury (RI) or irreversible injury (II) by measuring cytochrome c oxidase activity. Activity measured in the presence of DDM which destroys mitochondrial outer membranes, was taken as total cytochrome c oxidase activity as detailed in Experimental Procedures. Compared with control group, reversible calcium treatment (RI) induced injury of about 15% of mitochondria. In contrast, hypotonic treatment (II) caused more than 80% of mitochondria to burst. 2B. Measurement of cytochrome c release by immunoblotting. Irreversible injury (II) induced more cytochrome c release from mitochondria compared with reversible injury (RI). 2C. Determination of mitochondrial swelling. Mitochondrial swelling was determined in control group, where CsA did not have an effect; in the group of reversible injury (RI), where CsA prevented calcium-induced swelling; and in the group of irreversible injury (II), where CsA did not prevent calcium-induced swelling. 2D. The morphology of the isolated mitochondria as determined by electron microscopy. Left panel shows that control, non-treated mitochondria were a homogeneous population with intact membranes and orthodox cristae structure. The middle panel shows a similar picture of mitochondria after reversible injury (RI) with few injured/swollen mitochondria and moderately perturbed cristae structure. In contrast, irreversible injury (II) induced rupture of the majority of mitochondria with a marked absence of intact structure (right panel) (magnification 19,000×). 2E. Detection of oxidation as posttranslational modification of identified proteins by immunoblotting. Upper panel shows a stronger signal in irreversible injury, suggesting that irreversible injury was associated with the release of a greater level of ROS-modified proteins. Middle panel showed VDAC1 was modified by ROS in irreversible injury. Lower panel showed total VDAC1 protein expression as internal control.