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. 2016 Feb 4;35(7):724–742. doi: 10.15252/embj.201592862

Figure 4. mTORC1 integrates DDR signals towards mitochondrial biogenesis during cellular senescence.

Figure 4

  1. Representative Western blot of mTORC1 activity measured by phosphorylated p70S6K (T389) from 6 to 72 h after 20 Gy in MRC5 fibroblasts. Data are representative of three independent experiments.
  2. Representative Western blots of the mitochondrial proteins TOMM20, NDUFB8 (complex I), SDHA (complex II), UQCR2 (complex III) and MT‐CO1 (complex IV) following 20‐Gy irradiation with or without 100 nM rapamycin treatment in MRC5 fibroblasts. Data are representative of 4 independent experiments.
  3. Effect of 100 nM rapamycin on mitochondrial mass (measured by NAO fluorescence) 2–4 days following replication exhaustion (RS), genotoxic stress (generated by X‐ray irradiation, etoposide, neocarzinostatin (NCS), H2O2) or telomere dysfunction (TRF2ΔBΔM) in a variety of cell lines. Data are mean from 3 independent experiments per cell line or treatment.
  4. (top) Representative transmission electron microscopy (T.E.M.) micrographs of proliferating and senescent (3 days after 20‐Gy X‐ray) MRC5 fibroblasts treated with or without 100 nM rapamycin. Mitochondria are labelled in pink. Scale bar = 2 μm; (bottom left and middle) Quantification of mitochondrial volume fraction (%Vv) and mitochondrial number per cross‐section in proliferating and senescent (3 days after 20‐Gy X‐ray) MRC5 fibroblasts treated with or without 100 nM rapamycin. T.E.M. mitochondrial analysis is mean ± SEM of 24 electron micrographs per condition; (bottom right) mtDNA copy number analysis by qPCR in proliferating and senescent (3 days after 20‐Gy X‐ray) MRC5 fibroblasts treated with or without 100 nM rapamycin. Data are mean ± SEM of n = 3 independent experiments; asterisks denote a statistical significance at P < 0.05 using one‐way ANOVA.
  5. Representative Western blots showing the expression of phosphorylated p70S6K (T389) and AKT (S473), the mitochondrial protein NDUFB8 and the DDR downstream target p21 in MRC5 fibroblasts treated with or without 10 μM of the ATM inhibitor KU55933 and in fibroblasts from a patient with ataxia telangiectasis (AT) at different time points after 20‐Gy X‐ray. Data are representative of three independent experiments (ATM inhibitor) and 1 experiment (AT patient).
  6. Western blots showing the effect of the ATM inhibitor KU55933 on γH2A.X, AKT phosphorylation and p21 expression in MRC5 fibroblasts after 20‐Gy X‐ray. Data are from one experiment.
  7. Effect of rapamycin and/or ATM inhibitor (KU55933) on mitochondrial mass (NAO intensity) in proliferating and senescent (3 days after 20‐Gy X‐ray) MRC5 fibroblasts. Data are mean ± SEM of n = 3 independent experiments. Asterisks denote a statistical significance at P < 0.05 using one‐way ANOVA.