A. Mitochondrial fractions M1, M2 and M3 are shown in the 30% Percoll gradient. B. ROS was measured with the H₂O₂-sensitive dye amplex red (10 μM). Arrows mark the addition of cardiac mitochondria, crude, M1, M2 and M3 fractions (250 μg), and succinate (3 mM) to the reaction media. M3 but not M2 or M1 mitochondrial fractions produced normal amount of ROS as the crude mitochondria. C. Western blot analysis of whole heart lysate (WHL), crude mitochondria (CM), M1, M2 and M3 fractions. Antibodies and type of protein markers are given in each blot. Equal amounts (50 μg) of proteins were loaded on each lane. D. Quantification of Western blots normalized to the Ponceau S signal in each experiment (n=3 except for VDAC1, n=5 and Cox2, n=7). M3 mitochondrial fraction was enriched with VDAC1 and Cox2, and was practically devoid of other organelle markers including plasma membrane, Golgi, endoplasmic reticulum, T-tubules and nucleus. *, M3 significantly different from WHL; ^# M3 significantly different from CM (p<0.05).

Purified M3 fraction mitochondria were loaded with mitotracker and counter-stained for Cox2, VDAC1 and Lamin b1 proteins. A-B. Anti-Cox2 monoclonal antibody and mitotracker signals. C. Overlay shows high correlation between mitotracker and Cox2 signals. D-E. Anti-VDAC1 monoclonal antibody and mitotracker signals. F. Overlay of D and E shows high signal correlation of mitotracker with VDAC1. A'-F'. Squares in A-F are shown at higher magnification for better examination. G-H. Lamin b1 (nuclear envelope marker, negative control) and mitotracker signals. I. Anti-Lamin b1 polyclonal antibody and mitotracker signals do not coincide; thus, contaminant nuclear envelope fragments can be clearly distinguished from mitotracker-loaded mitochondria. G'-I'. Squares in G-I are shown at higher magnification for better examination. J-K. Mitochondria were loaded with mitotracker and labeled with secondary antibody alone. Secondary antibody (anti-mouse) showed no specific signals (J), while mitotracker labeling was robust (K) confirming the presence of mitochondria. L. Protein Proximity Index (Wu et al., 2010) of mitotracker towards Cox2, VDAC1, Lamin b1, or secondary antibody alone (Sec Ab) (black bars) and vice versa (open bars) (n=5 mitochondria preparations). Values are given in the text. Confocal images were acquired at 0.0575 μm/pixel.

Purified M3 mitochondria were labeled with monoclonal antibodies directed against Cox2 and VDAC1. A. Confocal image of Cox2 labeled mitochondria. B. Corresponding STED image with clear clusters. C. Overlay of A and B highlights increased resolution with STED. D. Square in C was magnified for better visualization. E. Magnification of square in B. F. Intensity profile of the line in E. Arrows mark an intensity peaks with FWHM of ~20 nm and ~33 nm. G. Cluster size frequency histogram for Cox2 was manually fitted to the sum of two Gaussian distributions; cluster size peaks were at 20 and 29 nm. H-I. Confocal and STED images of mitochondria labeled for VDAC1. J. Overlay. K. Square in J was magnified highlighting increased resolution of STED image and VDAC1 clusters. L. Square in I at higher magnification. M. Intensity profile of line scan in L. Arrow marks a peak with FWHM of ~33 nm. N. Cluster size frequency histogram for VDAC1 was manually fitted to the sum of four Gaussian distributions; cluster size peaks were at 22, 33, 55, and 83 nm. n=3 independent mitochondria isolations.