Figure 1.
DDR is detectable months after establishment of telomere-initiated cellular senescence.
a. In BJ cells, DDR, in the form of γH2AX foci, is detectable long time (up to three months) after establishment of telomere-initiated cellular senescence. Scale bar, 40 µm. b. Bar graph shows the fraction of γH2AX foci-positive cells ± s.e.m. before (young and pre-sen) and at the indicated time points after senescence establishment. Cells were considered positive if bearing more than 3 DDR foci (*** p-value <0.001). c. Bar graph shows the average number of γH2AX foci ± s.e.m. per cell at the indicated time points before and after senescence establishment (*** p-value <0.001; * p-value <0.05).
Figure 2.
Telomere-initiated senescent cells retain active DDR foci for years after senescence establishment.
a. DDR, in the form of ATM pS1981 foci co-localizing with 53BP1 and γH2AX foci, is detectable three years after senescence establishment. Scale bar, 10 µm. Below, bar graphs show the percentage of cells positive ± s.e.m. for the indicated DDR markers, in senescent (sen), early passage proliferating (prol) or telomerized proliferating (tel) skin fibroblasts from two independent centenarian donors (cen2 and cen3). Cells were considered positive if bearing more than 3 DDR foci (*** p-value <0.001). b. SA-β-gal staining of the two batches, cen2 and cen3, is shown together with the percentage of BrdU-positive cells. Scale bar, 100 µm.
Figure 3.
Unstable cellular senescent state is associated with loss of DDR foci.
a. Bar graphs show the average number of γH2AX foci ± s.e.m. per cell before (young and pre-sen) and at the indicated time points after senescence establishment in two different types of fibroblast cell strains, BJ and WI-38. b. Bar graphs show the fraction of γH2AX foci-positive cells ± s.e.m. at the indicated time points after senescence establishment in BJ vs WI-38. Cells were considered foci-positive if the number of foci were more than three (*** p-value <0.001). c. WI-38 senescent cells (WI-38 sen) in culture tend to reduce in number with time (day 30 and day 60) whereas BJ senescent cells (BJ sen) do not. The graph shows the fold change in cell number ± s.d. normalized to the number of senescent cells plated at day 0 (** p-value <0.01).
Figure 4.
Apparent differential DNA damage response activation during IR-induced cellular senescence correlates with cell survival.
a–b. BJ and IMR-90 cells were irradiated with 20 Gy and stained for 53BP1, as a DDR marker, three, ten and thirty days later. Bar graphs show the quantification of 53BP1-positive cells and number of 53BP1 foci per cell ± s.e.m. Cells were considered positive if bearing more than 3 DDR foci (*** p-value <0.001). c. BJ and IMR-90 cells were irradiated or not with 20 Gy. Graphs show the average cell number ± s.d. at different time points, in irradiated cells and non-irradiated controls (* p-value <0.05; ** p-value <0.01).