Ve 821

U2OS and HEK293T cell lines were purchased from ATCC. U2OS‐based EJ5 and DR‐GFP cell lines were gifts from Dr. Jeremy Stark (City of Hope, Duarte, CA, USA) and Dr. Xiaofan Wang (Duke University, Durham, NC, USA). All cell lines were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) in an atmosphere of 5% CO₂ at 37°C. For real‐time WRN recruitment studies, 1 × 10⁶ cells were transfected with 1 µg of pcDNA3.1‐mCherry‐WRN plasmid using Amaxa Cell Line Nucleofector Kit V (Lonza, Basel, Switzerland) by following the company's transfection protocol. For inhibiting the activities of DNA‐PKcs, ATM, ATR, CDK1, and CDK2, the cells were, respectively, treated with NU7026 (Tocris, Bristol, United Kingdom), KU55933 (Tocris), VE821 (Tocris), RO3306 (Tocris), or CDK2 inhibitor 2‐III (EMD Millipore, Burlington, MA, USA) for 2 to 4 h, as stated in the legend. To achieve ectopic expression of 3×FLAG‐WRN, 1 × 10⁷ 293T cells were transfected with 5 µg of pcDNA3.1 carrying 3×FLAG or 3×FLAG‐WRN using JetPrime transfection reagent (Polyplus Transfections, New York, NY, USA).

NU5455 and NU7441 were synthesized by the Newcastle University Medicinal Chemistry Department or NewChem Technologies Ltd. Full synthesis details for NU5455 are provided in patent WO 2010/136778 (example 102; compound 143). NU5455 was dissolved in DMSO for in vitro work, and prepared in N-methyl-2-pyrrolidone (NMP)/30% encapsin/PEG400 (1:6:3 vol/vol/vol) or 1% acetic acid (vol/vol) for oral administration in vivo. NU7441 was synthesized as detailed by Leahy et al. (51 (link)). KU55933, rucaparib, and VE-821 were purchased from Tocris Bioscience. All other reagents were purchased from Sigma-Aldrich unless otherwise stated. All compounds were dissolved in anhydrous DMSO and stored in small aliquots at –20°C, unless otherwise stated. Doxorubicin solutions were prepared in sterile, molecular-grade water and stored under light-protected conditions at 4°C.