Fig 2: DSB repair improvement by acceptor–donor cell interaction depends on ATM kinase activity. a Residual, 24–h 53BP1 foci in ATMwt fibroblasts (acceptor) pretreated with DNA–PK or ATM inhibitors, irradiated with 6–Gy x–rays and subsequently cultured alone or in co–culture with untreated MiaPaCa–2 cells (donor). DMSO used as control. b Residual, 24–h 53BP1 foci numbers in 6–Gy irradiated ATMwt or ATM-/- fibroblasts (acceptor) grown in mono– or co–culture with untreated MiaPaCa–2 cells (donor). c Normalized residual 53BP1 foci numbers in 6–Gy irradiated MiaPaCa–2 cells (acceptor) in mono– or co–culture with either ATMwt or ATM-/- fibroblasts (donor) or ATMwt fibroblasts pretreated with ATM inhibitor (DMSO used as control). d Comparison of 53BP1 foci numbers in 6–Gy irradiated MiaPaCa–2 cells (acceptor) cultured either alone or in co–culture with untreated MiaPaCa–2 cells, Capan1 cells, ATMwt or ATM-/- fibroblasts (donors). Number of analysed irradiated acceptor cells is n = 30, unless otherwise indicated. Data represent mean ± SD (two–sided t–test; ns, not significant, **P < 0.01, ***P < 0.005)

Fig 3: Activated ATM is predominantly responsible for signaling events during MVM infection.(A) ATM inhibitor reduces signaling events. Western blot analysis comparing DDR proteins at 18 h.p.i. of parasynchronized A9 cells infected with MVMp in the absence or presence of 7.5 µM ATM inhibitor KU 55933 (ATMi). (B) ATM-mediated signaling events seen in the absence of DNA-PKcs. Western blot analysis of DDR proteins following mock or MVMp infection of CHO-AA8 and CHO-V3 cells deficient in DNA-PKcs. Treatment with 10 µM ATM inhibitor KU 55933 (ATMi) blocked signaling in the CHO-V3 cells. (C) Activation of ATM following MVM infection. Western blot analysis shows ATM phosphorylation on S1981 following MVM infection of NB324K cells and hydroxyurea (HU) treatment. (D) ATM-dependent staining at APAR bodies. NB324K cells were infected with MVMp (MOI of 10) for 16 hr before being fixed and processed for immunofluorescence. Cells were stained with antibodies to NS1 and an antibody generated to phosphorylated ATM. Nuclei were stained with DAPI. All images were captured using an objective of 63×.

Fig 4: Oxidative stress-induced cellular senescence is reduced by genetic depletion of Atm. (A) Proliferation of WT (black), Ercc1-/- (blue) and Ercc1-/-Atm+/- (red) MEFs serially passaged at 20% oxygen was measured by an automated cell counter. Data shown are representative of three independent experiments using distinct MEF lines. (B) SA-ßgal staining of serially passaged MEFs cultured at 20% oxygen. Shown are representative images of passage 5 WT, Ercc1-/- and Ercc1-/-Atm+/- MEFs taken at 10x magnification. (C) The average percentage of SA-ßgal positive cells at each indicated passage. Ten fields were acquired and quantified per sample. Data shown are representative of two independent experiments. (D) Senescent WT, Ercc1-/- and Ercc1-/-Atm+/- MEFs (passage 5) cultured at 20% oxygen for 72 hrs were collected and lysed for immunoblot analysis of p16INK4a. (E) Supernatant collected from senescent WT, Ercc1-/- and Ercc1-/-Atm+/- MEFs was analyzed by ELISA for secreted IL-6. Graphs represent mean+/- s.e.m. P value was determined using Student’s t-test. *p<0.05, **p<0.01.

Fig 5: Genetic reduction of Atm improves bone and intervertebral disc pathology in progeroid Ercc1-/? mice. (A) Representative micro-CT images of lumber spines comparing severity of osteoporosis in 16-week-old WT, Ercc1-/?, Ercc1-/?Atm+/- mice. n=3-5 per group. Quantification of vertebral porosity, trabecular number, trabecular separation, thickness of trabecular bone was performed and shown. (B) Safranin O staining for disc matrix in thoracic discs from 12-week-old Ercc1-/? and Ercc1-/?Atm+/- mice. (C) GAG content measured by DMMB assays with NP tissues isolated from 12-week-old lumber discs. n=3 each group. Mean+/- s.e.m. P value was determined using Student’s t-test. **p<0.01.