Mitochondria are required for pro-ageing features of the senescent phenotype (2016)
Figure 6

Figure 6 - mTORC1-PGC-1β regulate mitochondrial content and contribute to senescence in vivo.

A) (top) Representative image of immunofluorescence double staining for the mitochondrial protein MT-CO1 and the DDR marker γH2A.X (scale bar=5µm) and (bottom) quantification of MT-CO1 intensity vs number of γH2A.X foci in hepatocytes from 12 months old mice. Data are mean±S.E.M of n=3 mice (at least 30 cells were analyzed per mouse); Asterisks denote statistical significant P<0.05 using Two-tailed t-test.

B) Representative westerns blots of pS6, S6, p21, PGC-1β, MT-CO1 and NDUFB8 protein expression in mouse liver tissue at 3 and 12 months of age. Data are from n=3 mice per group;

C) Effect of 4 months of rapamycin supplemented diet on PGC-1β expression in liver tissue of 16 months old mice. Data are from n=3 mice per group;

D) (top) Quantification of mitochondrial number per cross section and mitochondrial volume fraction (%Vv) in hepatocytes from 16 months old mice with or without 4 months rapamycin diet. Data are mean±S.E.M of n=3 mice per group (at least 20 cells were analyzed per mouse); (bottom) Representative electron micrographs of hepatocytes from 16 months old mice with or without 4 months rapamycin diet, (mitochondria are labelled in pink). Scale bar=5µm;

E) mtDNA copy number (measured by q-PCR) in mice liver tissue at 3, 12, 16 months and at 16 months after 4 months rapamycin diet. Data are mean±S.E.M of n=3-4 mice per group; Asterisks denote statistical significant P<0.05 using One-way ANOVA.

F) Oxygen consumption rates (OCR) in isolated liver mitochondria from 16 months old mice fed with or without rapamycin for 4 months, in the presence of pyruvate/malate. State III was induced by injection of ADP. State IV was induced by inhibition of the ATP synthase with oligomycin and uncoupled respiration rates were determined by injection of FCCP. Antimycin A (AA) was used to determine background, non-mitochondrial OXPHOS, OCR. Data are mean±S.E.M of n=5 mice per group;

G) (top) Representative immuno-TeloFISH images of hepatocytes from 3 and 15 months old mice with or without rapamycin (12 months diet). Co-localizing foci are amplified in the right panel (amplified images are from single Z-planes where co-localization was found); (bottom) Dot plot graph of Telomere-associated foci (TAF) in 3, 15 and 16 months old mice (15 and 16 months old mice were fed with rapamycin for 12 and 4 months respectively). Data are from n=3-9 mice per group (at least 50 cells were analyzed per mice);

H) (top) 4 months old and 15 months old mice livers [control (-) or rapamycin (+)] stained with Sen-β-Gal solution (Sen-β-Gal activity is evidenced by blue staining). Data are from n=3 mice per group; (bottom) Representative image showing Sen-β-Gal staining (Sen-β-Gal activity is evidenced by blue staining) in hepatocytes and corresponding immuno-TeloFISH (arrows represent co-localizing foci); Asterisks denote statistical significant P<0.05 using One-way ANOVA.

I) Representative Western blot showing the effect of 4 months rapamycin feeding on p21 expression in 16 months old mice. Data are from n=3 mice per group;

j) Effect of 4 months rapamycin feeding on mRNA expression of the SASP components CXCL1, CXCL5 and Inhibin A in liver tissue of 16 months old mice with or without 4 months rapamycin treatment. Data are from n=5 mice per condition; Asterisks denote statistical significant P<0.05 using Two-tailed t-test.

K) Mean number of TAF in hepatocytes of wild type and PGC-1β -/- mice with 18 months of age. Data are mean±S.E.M of n=4 mice per group; Asterisks denote statistical significant P<0.05 using Two-tailed t-test.

L) Scheme represents overall hypothesis: feedback loop between DDR, mTORC1 and mitochondrial biogenesis stabilize cellular senescence and are key factors for the development of the senescence-associated pro-oxidant and pro-inflammatory phenotypes.