Cdk2 inhibitor 2 3

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).

Generation of laser-induced DSB and recruitment of fluorescence protein-tagged proteins were performed with a SRS NL100 nitrogen MicroPoint system equipped to a Nikon Eclipse TE2000 spinning disk confocal microscope with Volocity software 6.3 (Perkin-Elmer)^{34 (link),64 (link)}. For treatment of CDK inhibitors, U2OS cells expressing GFP/YFP-tagged RECQL4 and YFP-MRE11 were pre-incubated with DMSO, 20 µM Roscovitine (Santa Cruz), 10 µM CDK1 inhibitor RO3306, 10 µM CDK2 inhibitor 2-III (EMD Millipore) for 4 h under standard cell culture conditions, and then subjected to micro point laser-irradiation. To study the function of CDK-dependent phosphorylation of Ser89 and Ser251 on RECQL4 recruitment to DSBs, GFP-tagged WT RECQL4, RQ4-2A, and RQ4-2D were expressed by transfecting the plasmids pEGFP-C1-RQ4Wt, pEGFP-C1-RQ4-2A, and pEGFP-C1-RQ4-2D into U2OS cells, respectively. For CDK 1and CDK2 inhibition, the U2OS cells were incubated with RO3306 and CDK2 inhibitor-III for 4 h before laser irradiation. The recruitment of these proteins was conducted as described above. The fluorescence intensity of the damaged area was measured with Volocity imaging software and normalized to that of a control area. The results are presented as mean ± s.e.m., and P -values were measured with Student’s t-test.