Fig. 4. The mitochondrial permeability transition contributes to mitochondrial swelling caused by overexpression of 1-deleted hFis1. (A) Clone 9 cells were transfected with Myc-hFis1[32-152] alone or co-transfected with Bcl-2 and Myc-hFis1[32-152], and scored for the swollen mitochondrial morphology. Co-expression of Bcl-2 with Myc-hFis1[32-152] reduced the number of cells containing swollen mitochondria by more than twofold, indicating that the mitochondrial permeability transition (MPT) played a role in mitochondrial swelling. Incubating cells transfected with Myc-hFis1[32-152] with MPT inhibitors bongkrekic acid (BA) or cyclosporin A (CsA) also decreased mitochondrial swelling. (B) Cobalt-quenched calcein measurement. Clone 9 cells transfected with Myc-hFis1[32-152] were loaded with calcein and CoCl₂ and stained with MitoTracker Red. (Left panel: mito) Swollen mitochondria by overexpression of Myc-hFis1[32-152] were visible in cells with asterisks. (Right panel: calcein) Calcein fluorescence was greatly reduced in swollen mitochondria (cells with asterisks). Cobalt ions entered mitochondria and quenched calcein fluorescence, indicating that the MPT occurred in swollen mitochondria. (C) Quantitation of calcein fluorescence in mitochondria. Calcein fluorescence in the mitochondrial regions was quantified from digital images. Mitochondrial regions were identified based on MitoTracker staining. Because of cell-to-cell variations of calcein fluorescence, intensity values from each cell were plotted to show the distribution, and median values were indicated as arrows. Fluorescence intensity was measured in 43 cells each for untransfected (UT) and transfected ([32-152]) at 24 hours post transfection (black circle and triangle, respectively), and in 28 cells each for UT and [32-152] at 48 hours post transfection (gray symbols). Experiments were repeated three times and similar quantitative results were obtained in all three experiments.