Fig. S1. (A) HCA2 cells were irradiated with 0.5 Gy. At the indicated intervals thereafter, they were stained for 53BP1, as in Fig. 1A. Cells were scored for the percentage of nuclei with three or more 53BP1 foci (red line). Shown are the means ± s.d. from three or more experiments. (B) Cells were untreated (‘Control’) or irradiated with 10 Gy. At the indicated intervals thereafter, they were fixed and stained for 53BP1 (red) and phosphorylated ATM or ATR serine/threonine substrate motifs (p-STK-SUB, green). (C) Cells were infected with lentivirus encoding p16^INK4A. Thirty hours later, cells were pulsed with BrdU for 18 hours, fixed and stained for p16^INK4A (red) and DNA synthesis (BrdU incorporation, green). DNA was counterstained with DAPI (blue). p16^INK4A-positive cells do not incorporate BrdU (white arrows). (D) Cells were infected with the p16^INK4A lentivirus. Forty-eight hours later, they were irradiated (10 Gy, XRA) or mock irradiated (‘Control’); 5 days thereafter, they were stained for 53BP1 (red) and p16^INK4A (green). DNA was counterstained with DAPI (blue). Lower panels display the red channel (53BP1 staining) in grayscale to facilitate visualizing damage foci. 53BP1 foci were present in irradiated p16^INK4A positive cells (white arrows). (E) HCA2 cells expressing MDC1−eGFP were irradiated with 10 Gy. Eight days later, MDC1−eGFP-positive cells in selected fields were imaged for a period of 6 hours. Individual cells in the displayed field are labeled 1−4. The left panel shows cells 8 days after irradiation, and the right panel shows the same cells 6 hours later. Selected MDC1 DNA-SCARS in cell 2 are highlighted with color-coded triangles to facilitate visualization between the panels. Note that cells 3 and 4 moved substantially during the 6-hour period but still harbor the same MDC1 DNA-SCARS. (F) p53 expression was stably reconstituted in p53-deficient H1299 cells (Wang et al., 1999) using a retroviral vector (MSCVpuro-P53). The p53-deficient and p53-reconstituted cells were treated with 10 g/ml bleomycin for 2 hours. Twenty four hours later, they were fixed and stained using antibodies against 53BP1 (red), p53 (P53, green) or p53 phosphorylated at serine 15 (P53-SER15, green). Nuclei were counterstained with DAPI (blue). (G) HCT116 cells that express CHK2 (‘Parental’) and were CHK2 deficient (CHK2 KO) (Jallepalli et al., 2003) were irradiated with 12 Gy. One hour later, they were fixed and stained for 53BP1 (red) or CHK2 phosphorylated at threonine 68 (CHK2-T68, green) (Cell Signaling antibody #2661, lot#7). Nuclei were counterstained with Dapi (blue). (H) HCA2 cells were either untreated (‘Control’) or irradiated with 10 Gy. At the indicated intervals thereafter, they were fixed and stained for 53BP1 (red) and CHK2-pT68 (green) (Cell Signaling monoclonal antibody #2197). Yellow indicates merged red and green signals.

Fig S2. (A) Replicatively senescent (SEN-REP) HCA2 cells were stained for 53BP1 (red) and PML (green) and imaged by confocal microscopy. Yellow indicates merged red and green signals. (B) Cells given bleomycin (20 g/ml, 2 hours), then fixed and stained 12 days later, as in A, and imaged by confocal microscopy.

Fig S3. Additional images showing lung alveoli from mice 7 days after receiving 8 Gy ionizing radiation, analyzed as described in Fig. 7C−D. Nuclei were counterstained with DAPI (blue). 53BP1 is shown in red, and PML is shown in green. The tissue section is shown at ×2 and ×20 magnification. The large red boxed areas in the ×20 image delineate fields selected for 53BP1 foci scoring in Fig. 7.

Fig S4. Additional images showing lung bronchioles from unirradiated or irradiated (8 Gy) mice, as described in Fig. 7D−E. Nuclei were counterstained with DAPI (blue). 53BP1 is shown in red, and PML is shown in green. (A) Bronchiolar epithelial cells from an unirradiated or irradiated mouse, fixed 48 hours after ionizing radiation treatment. Red arrows highlight 53BP1 DNA damage foci in irradiated bronchiolar epithelial cells, which are not detected in control non-irradiated lungs. The image is at ×20 magnification. (B) Section from a lung, 7 days after irradiation, at ×20 magnification, centered on a bronchiole (top panel). The lower panel shows a different section acquired using confocal microscopy centered on the transition between the bronchiolar epithelial cells and underlying alveolar cells. The orange labels in the top panel show the position of alveolar and bronchiolar cells analyzed in Fig. 7. The white arrows in the lower panel indicate lung cells with DNA-SCARS.

Fig S5. (A) Tissue section of the liver from an irradiated mouse 7 days after receiving 8 Gy ionizing radiation. DNA, 53BP1 and PML staining is as described in supplementary material Figs S3−S4. The section is shown at ×4 and ×20 magnification. The large white boxed area in the ×20 image represents a field selected for 53BP1 foci scoring in Fig. 7. (B) Quantification of 53BP1 foci in the liver up to 14 weeks following irradiation. Cells were scored for the percentage containing one or more 53BP1 foci. Shown are the means ± s.d. from three or more independent measurements. (C) Summary of quantification of 53BP1 damage foci in all tissues analyzed up to 7 days following irradiation. (D) The capacity of the 53BP1 antibody (red) to detect DNA damage foci specifically was tested in mouse tissues by co-staining for the DNA damage chromatin mark γH2AX (green) and visualizing by confocal microscopy. Nuclei were counterstained with DAPI (blue). Sections from brain tissue from animals 48 hours after receiving 8 Gy ionizing radiation. 53BP1 and γH2AX showed extensive colocalization, indicating that the 53BP1 antibody recognized damaged chromatin in tissues.