Fig 1.
High dose of ionizing radiation induces cellular senescence.
(A) Quantification of mean number of SA-β-Gal positive cells in A375, MRC5 and 92–1 cells at various time points post X-ray radiation (10 Gy). (B) Quantification of mean number of SA-β-Gal positive cells in 92–1 cells at various time points post-irradiation. (C) Quantification of mean number of Ki67 positive cells in 92–1 cells at indicated time points post-irradiation. (D) Quantification of mean number of EdU labeled cells in 92–1 cells at indicated time points post X-ray radiation (10 Gy). (E) Up: The protein levels of p53 and p21 expression measured by western blotting, indicating persistent p53/p21 pathway activation after ionizing radiation treatment. OCM-1 was a positive control for p53. Down: The protein levels of p16INK4a, pRB and RB expression measured by western blotting. Data are mean ± s.e.m. (n = 3). Ctrl: non-irradiated samples. β-actin: loading control. (F) Representative micrographs of F-actin fluorescence stained 92–1 cells. Scale bar: 20μm. F-actin was stained with phalloidine-FITC.
Fig 2.
High LET radiation is more efficient to induce cellular senescence than low LET radiation.
(A) Quantification of mean number of SA-β-Gal positive cells and (B) Ki67 positive cells in 92–1 cells on the 5th day post treated with 0.5, 1, 3, 5, 7, 10 and 15 Gy of X-rays, carbon ions or iron ions. Data are mean ± s.e.m. (n = 3). (C) Quantification of mean number of EdU positive cells in 92–1 cells on the 5th day post treated with 0.5, 1, 3, 5, 7 and 10 Gy of X-rays. (D) Representative micrographs of EdU labeled 92–1 cells on the 5th day post treated with indicated dose of X-rays. Scale bar: 20 μm.
Fig 3.
High dose of ionizing radiation induces persistent DDR activation in 92–1 cells.
(A, B) Micrographs of DDR foci in 92–1 cells following exposure to 10 Gy of X-rays. Persistent DDR activations are detectable even on the 5th day post-irradiation in the form of pATM foci (A) and 53BP1 foci (B). Scale bar, 10 μm. (C) The fraction of 53BP1foci positive cells (± s.e.m.) and (D) the average number of 53BP1 foci per cell (± s.e.m.) at the indicated time points after irradiation. For the quantification analysis, 100 cells per time point were analyzed (*p<0.05). (E) Micrographs of apoptosis in irradiated 92–1 cells measured by Hochest33342/PI staining; Scale bar, 20mm. (F) Intracellular ROS levels measured by fluorescence microscopy after staining with the fluorescent probe DCF. Scar bar: 20μm.
Fig 4.
High LET radiation is more efficient to induce repair-resistant complex DNA damage.
(A) Representative micrographs showing recruitment and retention of 53BP1 and XRCC1 foci at the sites of damaged DNA induced by 5 Gy X-rays or iron ions in 92–1 cells. Scale bar: 10 μm. (B) Percentage of 53BP1 foci co-localized with XRCC1 foci at indicated times post-irradiation. Data are mean ± s.e.m. (n = 3). For the quantifications analysis, 50 cells were analyzed per time point. (C) The average number of 53BP1 foci per cell (± s.e.m.) at the indicated times post-irradiation. For the quantification analysis, 100 cells per time point were analyzed. (D) The number of repaired 53BP1 foci per cell at indicated times post-irradiation (the number of 53BP1 foci at 6 hours is defined as initial DNA damage). (*p<0.05).
Fig 5.
Low LET radiation induced persistent DDR foci are preferentially associated with telomeric DNA.
(A) Representative micrographs of co-localizations between 53BP1 foci and telomeric-PNA probes (Telo) in 92–1 cells, at the indicated time points after 5 Gy of X-ray exposure. Scale bar: 10 μm (1μm in TAF panels. TAF: Telomere-associated foci). The average number of 53BP1 foci per cell and the percentage of 53BP1 foci co-localized with telomeres in 92–1 cells exposed to 5 Gy of X-rays (B) or carbon ions (C). Data are mean ± s.e.m. (n = 3). For the quantification analysis, 120 cells were analyzed for 53BP1 foci experiments and 100 cells for co-localization experiments.
Fig 6.
A proposed cellular model with two different outcomes caused by low LET radiation or high LET radiation.
DD: DNA damage. TADD: Telomere-associated DNA damage.