Fig. 2

Cytoprotection by FP15. (A) Effect of peroxynitrite (50–600 µM) on mitochondrial respiration in RINm5F cells (left panel) and HU-VECs (right panel); concentration-dependent restoration of the suppression of mitochondrial respiration by FP15 (3–30 µM). *,** represent significant effects of FP15 in peroxynitrite-treated cells (p < 0.05, p < 0.01, respectively); n = 6 to 9. (B) Cytofluorometric analysis of thymocyte necrosis and apoptosis induced by peroxynitrite. Thymocytes were treated for 4 hr with 20–80 µM of peroxynitrite. Cells were then stained with Annexin V-FITC and propidium iodide (PI) and two-color analysis was performed by flow cytometry. During apoptosis, phosphatidylserine becomes exposed on the outer surface of the plasma membrane and can be detected by Annexin-FITC. Necrosis is characterized by the breakdown of plasma membrane integrity that can be measured by the uptake of the cell impermeable dye propidium iodide (5). An increase in the number of apoptotic (Annexin V-FITC single positive, bottom right quadrant) cells as well as necrotic (stained by both Annexin V-FITC and PI, top right quadrant) cells was observed in response to 20-µM peroxynitrite treatment, whereas necrotic cells dominate in response to 80-µM peroxynitrite. FP15-treated cells were protected against both necrotic and apoptotic cell death, and normal cells (bottom left quadrant) are predominant. Similar results were found in four independent experiments.