Figure 6.

Rapamycin persistently increases the number of mitochondria and produces a long-lasting rescue of altered mitochondria. (A) Representative pictures show total mitochondria stained with MTR-G from controls and following rapamycin treatment (10 nM) for 24 h and after rapamycin withdrawal (up to 14 d), Arrows indicate intensely fluorescent signals. (B) Graph reports the two-fold steady increase in mitochondrial MTR-G fluorescence induced by rapamycin, which persists unmodified for 14 days. Values are given as the mean ± SEM from N = 100 cells per group. (C) Representative pictures at TEM show the increase in mitochondria (M) counted directly as specific organelles. (D) Graph reports the count of mitochondria per cell at TEM which overlaps the two-fold increase measured at MTR-G fluorescence, which similarly persists at 14 days. Values are given as the mean±S.E.M from N = 50 cells per group. (E) Representative pictures depicting mitochondrial morphology evaluated based on the modified scale from Flameng et al. 1980 [23]. A score of 1 indicates that mitochondria possess broken crests (arrowheads) with ruptured mitochondrial membranes (arrows), and a spread matrix dilution (asterisk); a score of 2 indicates a mitochondrion that possess broken crests with spots of diluted matrix but membranes intact; a score of 3 indicates that mitochondria possess only spots of diluted matrix but intact crists and intact inner/outer membranes; the score of 4 indicates an intact mitochondrion. Graph (F) reports the percentage of altered mitochondria (including all kinds of alterations). Graph (G) reports the average of mitochondrial score. Values are given as (F) the percentage ± SEM from N = 50 cells per group or (G) the mean mitochondrial morphological score ± SEM calculated by averaging mitochondrial scores from at least 150 mitochondria from each experimental group. * p < 0.05 compared with controls. Scale bars: (A) = 18 μm; (C) = 260 nm; (E) = 230 nm.