U2OS Cell Lines for DNA Damage Analysis

U2OS 53BP1–GFP (Bekker-Jensen et al., 2005 (link)), U2OS NBS1–GFP (Lukas et al., 2004 (link); both a kind gift from Jiri Bartek), U2OS/TRE/I-SceI-19 (Lan et al., 2010 (link); a kind gift from Prof. A. Yasui) were grown in DMEM supplemented with 10% fetal bovine serum, 1% L-glutamine, plus 1% penicillin/streptomycin and G418 (250 µg/ml), Puromycine (1 µg/ml) or Zeocin (250 µg/ml), respectively. U2OS MDC1–GFP cells (Hable et al., 2012 (link), a kind gift from Guido Drexler) were grown in RPMI 1640, supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% penicillin/streptomycin and puromycin (0.8 µg/ml). KrasG12D; Trp53^−/−; Dicer1^f/− and KrasG12D; Trp53^−/−; Dicer1^−/− cells (Ravi et al., 2012 (link)) were grown in DMEM, 10% fetal bovine serum, 1% L-glutamine and 1% penicillin/streptomycin. DNA damage was generated by treatment with 50 ng/ml Neocarzinostatin (NCS; Sigma, N9162) for 20 min at 37°C. All cell lines are routinely screened against mycoplasma contamination.