Figure 1. TAF are persistent following X-ray-induced senescence in MRC5 fibroblasts with or without telomerase activity.

(a) Total number of γH2A.X foci in human fibroblasts 1 day following irradiation with 1, 5, 10 and 20 Gy. Data are mean±s.e.m. of n=15. (b) Total number of γH2A.X foci colocalizing with telomeres (TAF) in MRC5 fibroblasts 1 day after irradiation with 1, 5, 10 and 20 Gy. Data are mean±s.e.m. of n=15. (c) Representative images of γH2A.X immuno-FISH in MRC5 fibroblasts 2, 6 and 16 days after 20 Gy. Images are Huygens (SVI) deconvolved Z projections of 3-μm stacks taken with a ×100 oil objective. White arrows indicate colocalization, and colocalizing foci are amplified in the right panel (amplified images are from single Z planes where colocalization was found). Scale bar=10 μm. (d) Percentage of γH2A.X foci colocalizing with telomeres (%TAF) in MRC5 fibroblasts up to 26 days after irradiation with 20 Gy. Data are mean±s.e.m. of n=15. (e) Mean number of both TAF and non-TAF in MRC5 fibroblasts up to 26 days after irradiation with 20 Gy. Data are mean±s.e.m. of n=15. (f) Enrichment of γH2AX at sub-telomeric regions was determined on chromosome 12p by ChIP followed by real-time PCR with previously independently validated primers at indicated distances from the chromosome end (10 days irradiated compared with non-irradiated MRC5 fibroblasts). Graph shows log2 ratios of irradiated and non-irradiated samples. Each PCR reaction was performed in triplicate. Data are mean from two independent ChIP experiments. (g) Enrichment of γH2AX at telomere repeats by real-time PCR. Graph represents fold enrichment of γH2AX at telomeric repeats between non-irradiated and 10 days irradiated cells for two independent ChIP experiments. Red line indicates non-irradiated control. (h) Graphs represent quantification of γH2A.X and telomere signals in selected regions of interest (dotted lines from 1c).