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

Figure EV5. mTORC1 activation contributes to the senescent phenotype via PGC1‐β‐dependent mitochondrial biogenesis and ROS‐mediated activation of a DDR .

Figure EV5

  1. (left) Representative images of γH2A.X (red foci) immunofluorescence in MRC5 fibroblasts at different time points after 20‐Gy X‐ray with or without 100 nM rapamycin supplementation (scale bar = 5 μm) and (right) representative images of 53BP1 (green foci) immunofluorescence in MEFs, 3 days after 10‐Gy X‐ray with or without 100 nM rapamycin supplementation (scale bar = 5 μm).
  2. Effect of genomic damage‐ or oxidative stress‐induced senescence on the mean number (N) of γH2A.X foci in MRC5 fibroblasts with or without 100 nM rapamycin. Cells were analysed 3 days after the induction of senescence and treatment with rapamycin. Data are mean ± SEM of n = 3 independent experiments; asterisks denote a statistical significance at P < 0.05 using one‐way ANOVA.
  3. (top) Representative Western blot showing the inhibition of p21 protein expression with 100 nM rapamycin at different time points (days) after 20‐Gy X‐ray in MRC5 fibroblasts. Data are representative of three independent experiments; (bottom) p21 mRNA levels at 3 days after 20‐Gy X‐ray with or without 100 nM rapamycin treatment in MRC5 fibroblasts. Data are mean ± SEM of n = 3 independent experiments; asterisks denote a statistical significant at P < 0.05 using one‐way ANOVA.
  4. Western blots showing the knockdown efficiency of two different siRNAs against mTOR and its effects on p70S6K phosphorylation (T389), 2 days after 20‐Gy X‐ray in MRC5 fibroblasts. Data are representative of two independent experiments.
  5. Effect of 100 nM rapamycin supplementation on Sen‐β‐Gal activity in MRC5 fibroblasts induced to senescence following treatment with 20‐Gy X‐ray irradiation, 80 ng ml−1 neocarzinostatin (NCS), 50 μM etoposide, 400 μM H2O2 and replicative exhaustion (RS). Cells were analysed 10 days after the induction of senescence and treatment with rapamycin. Data are mean ± SEM of n = 3 independent experiments (at least 80 cells were analysed per condition). Asterisks denote a statistical significance at P < 0.05 using one‐way ANOVA.
  6. Quantification of Ki67‐positive MRC5 fibroblasts 3 and 10 days following 20‐Gy X‐ray irradiation with or without 100 nM rapamycin treatment. Data are mean ± SEM of n = 4 independent experiments; asterisks denote a statistical significance at P < 0.05 using one‐way ANOVA.
  7. Secreted protein array of a variety of inflammatory proteins following 20‐Gy X‐ray‐induced senescence in MRC5 fibroblasts with or without 100 nM rapamycin treatment (3 and 10 days after 20‐Gy X‐ray). Data are mean of three independent experiments.
  8. mRNA expression of IL‐6 after 20‐Gy X‐ray (left) with or without 100 nM rapamycin treatment in MRC5 fibroblasts and (right) in human fibroblasts from an AT patient. Data are mean ± SEM of n = 3 independent experiments (for MRC5+Rap cells) and mean ± SD of n = 2 independent experiments (for AT cells).
  9. Representative Western blots of mTORC1 activity, measured by p70S6K phosphorylation (T389) in wild‐type and PGC‐1β −/− MEFs overexpressing RhebN153T (1 day after 10‐Gy irradiation). Data are representative of three independent experiments.
  10. Effect of rapamycin supplementation on Sen‐β‐Gal activity in wild‐type and PGC‐1β −/− MEFs, 10 days after 10‐Gy X‐ray. Data are mean ± SEM of n = 3 independent experiments (at least 100 cells were analysed per condition). Asterisks denote a statistical significance at P < 0.05 using one‐way ANOVA.