Fig. 6.

Mitochondrial responsiveness declines with age.

(A) Immunofluorescence staining of NSC34 cells for either PDH (red) or TOM20 (green) represents MDVs. Arrows indicate MDVs with either PDH or TOM20 positive staining, while arrowheads indicate fragmented mitochondria with colocalized staining of PDH and TOM20. Scale bar, 5 μm. (B) Representative live cell confocal imaging of NSC34 cells showing MDVs (∼300 nm) with MitoTracker Red (red) were transported by YFP-STX17 (green) in 10-min time-lapse images. Arrows indicate MDVs with colocalization of YFP-STX17 (green) and MitoTracker Red (red). The arrowhead indicates aggregation of STX17 (green aggregation, >2 μm). Scale bar, 2 μm. (C-D) Representative TEM images showing isolated liver mitochondria from young and middle-aged hSOD1^WT(C) and hSOD1^G93A(D) transgenic mice. Black arrows indicate MDVs (70–150 nm), and arrowheads indicate autophagosomes (>500 nm). Scale bar, 500 nm. (E-F) Quantification of the ratio of mitochondria with MDVs to the total amount of liver mitochondria from hSOD1^WT(E) and hSOD1^G93A(F) transgenic mice. (G) Representative Western blot images of protein level of MDVs isolated from hSOD1^WT and hSOD1^G93A mice liver. (H–I) Quantification of the protein levels of MDVs (PDH, TOM20, and STX17) (n = 3, mean ± SEM. **p < 0.01).