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

Figure EV1. Mitochondria are key factors for the development of pro‐ageing features of cellular senescence.

Figure EV1

  1. Effects of CCCP treatment on mitochondrial depletion, immediately (left) and 16 days (right) after 48 h of 12.5 μM CCCP treatment on irradiated (IR) MRC5 fibroblasts. Cells were irradiated with 20‐Gy X‐ray and treated with 12.5 μM CCCP at day 2 after irradiation. After 48 h of CCCP treatment, cells were either immediately collected for analysis (4 days after IR) or were kept in culture with normal serum‐supplemented medium for 16 days and then harvested for analysis (20 days after IR). Western blots are representative of three independent experiments.
  2. T.E.M. images of senescent Parkin‐expressing MRC5 fibroblasts at 10 and 20 days after irradiation, pre‐treated or not with CCCP. Scale bar = 1 μm; red arrows denote mitochondria. Note: when conducting 3D‐EM of 20 days after IR, one cell appeared to have lost Parkin and did not show the depletion of mitochondria (indistinguishable from controls), while those where depletion was complete (the majority) were devoid of intact mitochondria.
  3. Levels of secreted IL‐6 and IL‐8 proteins (measured by ELISA) in proliferating and senescent (10 days after 20‐Gy X‐ray) control and Parkin‐expressing MRC5 fibroblasts with or without 12.5 μM CCCP treatment. Data are representative of two independent experiments.
  4. (left) Representative flow cytometry histogram of mitochondrial mass staining (NAO) in parental and rho0 143B osteosarcoma cells (data are representative of three independent experiments), (middle) quantification of ROS levels (DHE intensity) and (right) mRNA abundance of the SASP factor IL‐6 in parental and rho0 cells following 10‐Gy X‐ray. Data are mean ± SEM of n = 3 independent experiments; asterisks denote a significance by one‐way ANOVA at P < 0.05.
  5. Representative images of the proliferation marker Ki67 (representative of two independent experiments), quantification of population doublings (PD) and BrdU‐positive cells of proliferating and senescent (10 and/or 20 days after 20‐Gy X‐ray) control and Parkin‐expressing MRC5 fibroblasts with or without 12.5 μM CCCP treatment. Data are mean ± SEM of n = 3 independent experiments for PD analysis and mean ± SD of 10 random panes for BrdU analysis.
  6. Representative Western blots of mTORC1 activity, measured by p70S6K phosphorylation (T389), in senescent (10 days after 20‐Gy X‐ray) control (C) and Parkin‐expressing (P) MRC5 fibroblasts. Data are representative of 3 independent experiments.
  7. Representative Western blots showing the absence of mitochondrial proteins (NDUFB8, SDHA, UQCRC2 and TOMM20), expression of p21 and mTOR activity (measured by phosphorylation of the p70S6K (T389)) in proliferating control and Parkin‐expressing MRC5 fibroblasts (10 days after 48 h of 12.5 μM CCCP treatment). Data are representative of two independent experiments.
  8. Quantification of BrdU‐positive cells (data are mean ± SD of n = 2 independent experiments), Sen‐β‐Gal activity (data are mean ± SD from 10 random planes), IL‐6 secretion measured by ELISA (data are representative of two independent experiments) and ROS levels measured by DHE intensity (data are mean ± SD of n = 3 technical repeats) of proliferating control and Parkin‐expressing MRC5 fibroblasts (after 48 h of 12.5 μM CCCP treatment).
  9. Steady‐state cellular ATP levels were measured using an ATP luciferase Kit (Invitrogen) in proliferating control and Parkin‐expressing MRC5 fibroblasts (after 48 h of 12.5 μM CCCP treatment). Data are mean ± SEM of n = 3 independent experiments.