Figure 1. Mitochondrial Dysfunction and Retrograde Response in Senescent Cells.

(A) and (B) Confocal images of actively proliferating (PD 34) and senescent (PD 47) MRC-5 fibroblasts. (A) Red MitoSOX fluorescence indicates mitochondrial superoxide. Blue: DAPI. Note lower average red fluorescence and cell-to-cell heterogeneity in proliferating cultures. (B) JC-1 ratio images. A shift from red to green fluorescence indicates low MMP.

(C) MitoSOX, DHR, and JC-1 ratio intensities in senescent MRC-5 fibroblasts as measured by FACS (percentage of young fibroblast control, set to 100%). Data are mean ± s.e.m., n = 3. An asterisk (*) indicates a significant difference to young controls with p < 0.001.

(D) Integrity of mtDNA as measured by the relative amplification efficiency for an 11-kb mtDNA fragment in young and senescent MRC5 cells. Data are mean ± s.e.m. An asterisk (*) indicates a significant difference to young controls with p < 0.001.

(E) Single cell traces of cytoplasmic calcium levels [Ca²⁺]_i following a challenge with 100 mM CaCl₂ as measured by Fluo3 fluorescence confocal imaging.

(F) Basal cytoplasmic [Ca²⁺]_i levels and recovery times in young and senescent cells. Data are mean ± s.e.m. from 13 young and 8 senescent cells. An asterisk (*) indicates a significant difference between young and senescent cells with p < 0.001.

(G–J) Relative mRNA expression levels in young (YOU), senescent (SEN), and hyperoxia-treated (HYP) MRC5 (averages of two [HYP] to four [YOU] independent experiments per condition). Fold-change colour code is indicated on the bottom. For some genes, the arrays contained probe sets resulting in multiple occurrences. All genes showing at least a 2-fold expression difference between YOU and SEN were selected if the expression difference was confirmed in HYP and if they fell into one of the following categories: (G) Ca-dependent signalling; (H) glycolysis and Krebs cycle; (I) mitochondrial function and biogenesis; and (J) stress response and apoptosis.