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

Figure EV2. Alleviation of the senescent phenotype in mitochondria‐depleted cells does not compromise ATP generation.

Figure EV2

  1. Extracellular acidification rate (ECAR) of senescent (10 days after 20 Gy) control and Parkin‐expressing MRC5 fibroblasts with or without 12.5 μM CCCP treatment. ECAR was measured using a Seahorse XF24 analyzer. Data are representative graphs from the Seahorse XF24 analyzer software. Data are mean ± SD n = 4 technical repeats (representative of two independent experiments).
  2. ATP production rate in senescent (10 days after 20 Gy) control (C) and Parkin‐expressing (P) MRC5 fibroblasts with or without 12.5 μM CCCP treatment (calculated from the OCR and ECAR measured using a Seahorse XF24 analyzer). ATP production by mitochondria was calculated by multiplying the ATP turnover ((basal OCR) – (OCR with oligomycin) by the established phosphorus/oxygen (P/O) ratio of 2.3 (Brand, 2005). ATP production by glycolysis is considered to have a 1:1 ratio with lactate production. The extracellular acidification rate is mainly due to lactate and bicarbonate production and, when calibrated as the proton production rate, indicates glycolytic rate (Birket et al 2011; Wu et al, 2007). Data are mean ± SD, n = 4 technical repeats (representative of two independent experiments).
  3. Steady‐state cellular ATP levels were measured using an ATP luciferase assay (Invitrogen) in senescent control and Parkin‐expressing MRC5 fibroblasts with or without 12.5 μM CCCP treatment, at 10 days (data are mean ± SEM, n = 3 independent experiments) and 20 days (data are mean ± SD from n = 3 technical repeats) after 20‐Gy X‐ray. Asterisk denotes a statistical significance at P < 0.05 using one‐way ANOVA.
  4. Heatmap of the RNA‐seq analysis of glycolysis‐associated gene expression in proliferating and senescent (10 days after 20 Gy) Parkin‐expressing MRC5 fibroblasts with or without 12.5 μM CCCP treatment. Red indicates up‐regulated genes; green indicates down‐regulated genes. Data are from n = 3 independent experiments.