Af 1655

For the experiment with IR, cells were irradiated with 10 Gy and incubated for 1hr before harvesting. For the experiments with CPT, H₂O_2, and arsenite, cells were incubated with media containing 10 μM CPT, 100 μM H₂O_2, or arsenite for 1hr before harvesting. Cells were lysed in 10× cell lysis buffer (9803, Cell Signaling) and lysate (20 μg) was separated by 8% SDS-PAGE gel and analyzed by western blotting. Proteins were transferred to PVDF-FL membrane (Millipore) and probed with antibodies directed against ATM (sc-135663, Santa Cruz), phospho-ATM Ser-1981 (AF-1655, R&D Systems), KAP1 (ab22553, Abcam), phospho-KAP1 Ser-824 (A300-767A, Bethyl Laboratories), Chk2 (GTX70295, Genetex), and phospho-Chk2 Thr-68 (2661S, Cell Signaling) followed by detection with IRdye 800 anti-mouse (RL-610-132-121, Rockland) or Alexa Fluor 680 anti-rabbit (A21076, Invitrogen) secondary antibodies. Western blots were analyzed and quantitated using a Licor Odyssey system.

Using formalin fixed paraffin embedded (FFPE) tissues, HCC grading was by two pathologists (15 (link)). A detailed immunohistochemistry protocol is in supplementary methods. Briefly, antigen retrieval was with an Antigen Access Unit (A. Menarini diagnostics, Berkshire, UK). Antibodies: anti-DNA-PKcs (rabbit polycolonal, H-163; 1:500; Santa Cruz Biotechnology, Santa Cruz, CA), anti-phosphorylated Ser2056 DNA-PKcs (rabbit polyclonal, ab20407; 1:500; Abcam, Cambridge, UK), anti-ATM (rabbit polyclonal, MAT3-4G10/8; 1:800; Sigma, Poole, UK), anti-phosphorylated Ser1981 ATM (rabbit polyclonal, AF1655; 1:300; R&D Systems, Minneapolis, MN). Sections were analysed using Aperio^® Image analysis. Hepatocyte nuclei were identified using a modified nuclear algorithm and staining quantified in pixels after background subtraction. The selection of normal versus tumour areas was by a pathologist, while the application of the quantification algorithm was by supervised researchers. Both pathologists and researchers were blinded until the study endpoint.