Research Paper Volume 6, Issue 4 pp 296—310

Figure 5. Modulation of mitochondria fusion and fission processes. Cells were left untreated (white columns) or treated (black columns) for 4 hours with Mdivi-1 100 μM (A-C), or with CCCP 50 μM (D-F). (A,D) Semi-quantitative flow cytometric analysis of autophagy assessed with Cyto-ID Autophagy detection kit. In ordinate the median fluorescence intensity is reported. (B,E) Semi-quantitative cytofluorimetric analysis of mitochondrial mass assessed with MTG staining. In ordinate the percent increase in comparison with respective untreated controls is reported. (C) Densitometric analysis of western blotting of DRP1 over tubulin ratios. Results are expressed as mean value ± SD of 6 Young, 8 Old and 8 LLI. (F) Morphometric analysis of mitophagy by fluorescence microscopy. Cells are stained for TOM20 (indicating mitochondria, green fluorescence) and LAMP1 (indicating lysosomes, red fluorescence). We considered positive to mitophagy only those cells in which TOM20 and LAMP1 overlapped (yellow fluorescence) in up to five intracytoplasmic areas. (G-J) Two representative IVM micrographs showing mitochondria engulfed within lysosomes indicating mitophagy. In magnification of boxed areas (H and J panels, respectively) it is possible to appreciate TOM20/LAMP1 co-localization (yellow fluorescence areas, white arrows).