DNA Damage Response in Cell Lines

U2OS, HeLa, HEK293T, TOPORS^+/+ and TOPORS^−/− MEFs were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, USA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen). Cells were maintained in a humidified incubator containing 5% CO₂ at 37°C. All cells were obtained from the American Type Culture Collection (Manassas, VA, USA). TOPORS^+/+ and TOPORS^−/− mouse embryonic fibroblast (MEF) cells were obtained from Dr Eric H. Rubin (36 (link)). To induce DNA damage, exponentially growing cells were treated with ¹³⁷Cs γ-ray at a dose of 2 or 5 Gy (Gammacell 3000 Elan; Best Theratronics, Ottawa, Canada), 2 mM hydroxyurea (HU, Sigma-Aldrich, USA) and then allowed to recover at 37°C for indicated time points.