Atri ve 821

All cell lines were grown at 37 °C under standard cell culture conditions (humidified atmosphere, 5% CO₂). ALT-positive GM847, SW26, and U-2 OS cells, as well as derived cell lines, and ALT-negative HeLa, HeLa LT (long telomeres), SW39 and hTERT-RPE1 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (GIBCO) and penicillin-streptomycin antibiotics. For U-2 OS CycE/RAD52 WT and KO cells G418 400 μg/ml (Gibco, 10131-027), puromycin 1 μg/ml (Sigma, P8833) and tetracycline 2 μg/ml (Sigma, T7660) were added to the medium. U-2 OS 53BP1-GFP/RPA70-mScarlet cells were maintained in presence of puromycin 0.5 μg/ml (Sigma, P8833) and 5 μg/ml blasticidin (InvivoGen, ant-bl-05). All cells used in this study were grown under sterile conditions and routinely (monthly) tested for mycoplasma contamination and scored negative. The following compounds were used in this manuscript at the indicated final concentrations unless stated otherwise: ATRi Az-20 (5 μM for RPE-1 cells, else 1 μM, Tocris), ATRi VE-821 (5 μM, Selleckchem), APH (0,2 μM, Sigma-Aldrich), HU (2 mM, Sigma-Aldrich), Nocodazole (50 ng/ml, Sigma-Aldrich), CDKi RO-3306 (9 μM, Sigma-Aldrich), Colcemid (0,1 μg/ml, Roche), ATMi KU-55933 (10 μM, Selleckchem).

Colorectal cancer (CRC) cell lines (HCT116, HT29, WIDR and T87), lung cancer cell lines (A549, H460, H1299 and H1975), a human bronchial epithelial cell line (BEAS-2B), human glioma cell lines (U251, U373 and U87), human hepatoma cell lines (HuH7, 7721, HepG2) and human embryonic kidney 293 cells (HEK 293T) were all purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). The culture conditions used in this study are shown in Supplementary Table S1. High glucose Dulbecco’s modified Eagle’s medium (DMEM) and RPMI-1640 medium were supplied by HyClone (Thermo Fisher, USA). All culture media were supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher), 100 U/mL penicillin and 100 µg/mL phytomycin (Gibco, Thermo Fisher). All cultures were maintained at 37 °C and 5% CO₂. For induction of DNA damage, cells were treated with camptothecin (CPT, 1 µM, Selleck) for 1 h, etoposide (ETO, 100 μg/mL, Selleck) for 4 h, or γ-irradiation. ATM inhibitor (ATMi, KU-55933), ATR inhibitor (ATRi, VE-821) and DNA-PK inhibitor (DNA-PKi, NU7441) were purchased from Selleck. During DNA damage induction by ionizing radiation (IR), CPT or ETO, the corresponding inhibitors were maintained in complete medium continuously unless indicated.

The following drugs and checkpoint inhibitors were used: Hydroxyurea (Calbiochem), Aphidicolin (Acros Organics), Camptothecin (Calbiochem), ATRi VE-821 (Selleckchem), CHK1i LY2603618 (Selleckchem), ATMi KU-55933 (Selleckchem), DNA-PKcsi NU7441 (Selleckchem).