293h cells

HEK293 and U2OS cells were cultured in Dulbecco's-modified Eagle's medium, containing 4.5 g/l D-glucose and GlutaMAX (Life Technologies, Carlsbad, CA, USA), and supplemented with 10% fetal bovine serum. HEK293 cell culture medium also contained pyruvate. A FLAG-DNA-PKcs-expressing stable cell line was generated by transfecting a mammalian expression vector encoding FLAG-DNA-PKcs fusion protein (gift from Dr. David J. Chen, UTSouthwestern, Dallas, TX, USA) with pTKHyg plasmid into 293H cells (Life Technologies) and selecting individual clones with 200 μg/ml of hygromycin B (hygB). Individual hygB-resistant clones were screened for full-length FLAG-DNA-PKcs expression by western blotting. One clone expressing physiological levels of tagged-DNA-PKcs was selected for further experiments. Suspension-adapted HEK293 (HEK293S) cells were cultured in Freestyle 293 medium (Life Technologies) supplemented with hygB (100 μg/ml) between densities of 0.5–3 × 10⁶ cells per ml in conical flasks on a shaking platform (160 r.p.m.) in a humidified 37 °C incubator. HEK293S cells were transfected with pCMX-FLAG-XLS or pCMX-LacZ plasmid (gift from Dr. Thomas Perlmann, LICR, Stockholm, Sweden) using Freestyle MAX (Life Technologies) according to the manufacturer's instructions.

HEK293F cells were transfected using Freestyle MAX (Life Technologies) according to the manufacturer’s instructions or PEI (1.5 μg/ml). Stable HEK293 cell lines expressing FLAG-tagged PAXX, -XLF, and -XRCC4 were generated by co-transfecting mammalian expression vector encoding N-terminal FLAG-tagged proteins with pTKHyg plasmid into 293 H cells (Life Technologies) and selecting individual clones with hygromycin B (0.2 mg/ml). Individual hygromycin B-resistant clones were screened for expression of full length FLAG-tagged proteins by immunoblotting. A stable clonal HEK293 cell line expressing FLAG-tagged DNA-PKcs was used as described¹⁰ . HEK293F cells were cultured in Freestyle^TM 293 medium supplemented with hygromycin B (0.1 mg/ml) between densities of (0.5–3) × 10⁶ cells/ml in conical flasks on a shaking platform (160 rpm) in a humidified 37 °C incubator. RPE-1 cells were transiently transfected using Lipofectamine 2000 according to the manufacturer’s instructions (Life Technologies). Stable U2OS cell lines expressing N-terminal tagged EGFP- or mCherry-Pol λ or N-terminal tagged EGFP-FLAG-Ku70 fusion proteins were generated by transfection with PEI (2 μg/ml).