Figure 4: Telomere-associated foci can be visualized in live MEFs and are longer lived than non-telomeric foci.

(a) Percentage of 53BP1 foci colocalizing with telomeres (% TAF) in MEFs up to 10 days after irradiation with 10 Gy. (b) Mean number of both TAF and non-TAF in MEFs up to 10 days after irradiation with 20 Gy. (c) Representative images of Immuno-FISH using antibody against 53BP1 in fixed MEFs 1, 3 and 10 days after 10-Gy irradiation (red: telomeres, green: 53BP1). The white box indicates TAF and image on the right shows boxed image at higher magnification of a single Z plane. Images were taken with a Leica DM5500B fluorescence microscope. (d) Confocal time series of MEFs expressing AcGFP-53BP1c together with a PNA telomere probe 3 days after 10-Gy irradiation at the indicated times (min). Images are compressed stacks (maximum intensity projections) with a 4.5-μm focal depth. The white box indicates one TAF, which is permanent during the entire time series, and is shown at higher magnification on the right (single Z plane). White arrows indicate long-lived TAF and blue arrows short-lived non-TAF. (e) Kaplan–Mayer survival curves for AcGFP–53BP1c, which colocalize (TAF) or do not colocalize (non-TAF) with telomeres in MEFs 3 days after 10-Gy irradiation. The 200 foci were analysed from 8 cells. The Gehan–Breslow test shows a statistically significant difference between survival curves (P=<0.001). (f) Representative trace of AcGFP-53BP1c foci in one MEF for the indicated time 3 days after irradiation (10 Gy). Each bar represents the track of one individual focus recorded for the indicated time interval. 53BP1 foci colocalizing with telomere PNA probe are in red (TAF) and non-colocalizing foci are in black (non-TAF). A Zeiss LSM510 confocal microscope was used for live-cell imaging. Scale bars: 5 μm in d and 10 μm in c.