A simple stochastic step model explains Reactive Oxygen Species production in cell populations undergoing senescence and in immortal cells.

<p>A) Hypothesis: Schematic representation of a step increase in phenotype for 100 cells. Each coloured trace represents one cell as it undergoes transition from proliferation-competence to senescence. Phenotype for each cell in the proliferating state is drawn from a relatively narrow distribution; whereas the phenotype of senescent cells is drawn from a broader distribution, with a higher expected value. All phenotype transitions are strictly non-decreasing. With increasing PD a higher proportion of cells in the population become senescent. B) Normalised, mean MitoSOX fluorescence measured by flow cytometry varies with PD in MRC5 fibroblasts cultivated until senescence (blue points). Normalised mean mitochondrial mass in a distinct population of the same cell type varies with PD (red points). Linear interpolation approximations to these curves were constructed (red and blue lines) and MitoSOX/mitochondrial mass estimated (green line); C) Kinetics of interpolated (solid lines) normalised MitoSOX fluorescence (blue), normalised mitochondrial mass fluorescence (red) and MitoSOX/mitochondrial mass ratio (green) are compared with mean population estimates from a stochastic step model (dashed lines). Stochastic step model parameters are estimated from the distributions for a wholly proliferative population (PD₂₇) and for a wholly senescent population (PD₄₇).</p

Ki67 negative cells containing more than 5 γH2A.X foci have significantly higher MitoSOX levels than Ki67 positive cells.

<p>A) Representative fluorescent images of YOUNG (PD₃₀) and senescent (SEN, PD₄₇) cells stained with MitoSOX followed by immunostaining against Ki67 and γH2A.X. MitoSOX staining (red); Ki67 (yellow); γH2A.X (green); DAPI (blue). B) Boxplots showing MitoSOX intensity in Ki67+ fibroblasts at 3 different PDs (∼50 cells per condition were quantified) C) MitoSOX intensity in Ki67+, Ki67−<5 γH2A.X foci and Ki67−>5 γH2A.X foci MRC5 fibroblasts (∼50 cells per condition were quantified). Asterisk indicates significance when comparing Ki67−>5 foci with the two other groups by 2-Way ANOVA; D) Comparison between MitoSOX fluorescence kinetics obtained by flow cytometry and microscopy in human fibroblasts; E) MitoSOX data (grey area) and growth curve (red circles) of mouse ear fibroblasts grown under 20% oxygen (data are from 3 independent mice).</p

Mitochondrial ROS does not increase significantly with population doublings following <i>hTERT</i> overexpression.

<p>Mean MitoSOX fluorescence measured by flow cytometry at increasing PDs in a purely proliferative population of MRC5 fibroblasts (blue points), in the same population of cells undergoing transition to senescence (light blue points), and in two independent experiments using MRC5 fibroblasts immortalised by transfection with hTERT (green points & purple points). Fluorescence in MRC5 fibroblasts is normalised according to the value at PD₂₇, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032117#pone-0032117-g001" target="_blank">Fig. 1B</a>. All other datasets are normalised according to the fluorescence levels at the earliest observed PD.</p