Mitochondrial Dysfunction Accounts for the Stochastic Heterogeneity in Telomere-Dependent Senescence (original) (raw)
Figure 5
Cell-to-Cell Heterogeneity in Replicative Senescence: Involvement of Mitochondrial ROS and Telomeres
(A) The frequency of senescent cells as measured by γ-H2A.X (filled bars) or Sen-β-Gal (open bars) staining is time- and oxidative stress–dependent. Experiments started at PD 38, and cells were grown for the indicated time under either 21% oxygen (CONTROL), 40% normobaric oxygen (HYPEROXIA), normoxia + 250 μM DNP (DNP), 5% normobaric oxygen (HYPOX), or normoxia and 400 μM PBN (PBN). Data are mean ± s.e.m., n ≥ 3.
(B) Cells with high mitochondrial superoxide production are γ-H2AX positive. Cells were stained with MitoSOX, photographed, and then fixed and immunostained for γ-H2A.X; then the same area was photographed again and merged.
(C) ImmunoFISH in an actively growing MRC5 culture shows frequent co-localization (yellow) between γ-H2A.X foci (green) and telomeres (red) in those cells that do contain foci. This co-localisation is significant according to a Pearson correlation analysis (see Figure 6H).
(D) Telomere Q-FISH of metaphases from young (left and middle) and near-senescent (right) MRC5 cultures. A few metaphases from young cultures show weak telomeric signals (middle image) similar to typical metaphases from a near-senescent culture (right image).
(E) Cumulative Q-FISH telomere frequency histograms from a young (PD 13, top left) and a near-senescent (PD 43, top right) population together with histograms from two individual metaphases at PD 13 (bottom). Metaphases of the near-senescent type (bottom right) are found with low frequency (<10%) in young cultures.