Cells were grown in DMEM (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) at 37ºC/5% CO2. The stable HeLa cell line (mCherry-53BP1-FFR) used in Figure 1 was generated by transfecting a plasmid containing mCherry-53BP11220-1711 (Addgene, Cambridge, MA; Dimitrova et al., 2008 (link)) into a cell line stably expressing GFP-tubulin (Mackay et al., 2010a (link)) using Lipofectamine LTX (Life Technologies) and selecting with 0.5 mg/ml G418 plus 0.5 μg/ml puromycin. Generation of the HeLa cell line stably expressing GFP-tubulin and histone H2B-mCherry was previously described (Mackay et al., 2010a (link)).
To induce replication stress, cells were treated for 24 h with either 50 μM hydroxyurea (MP Biomedicals, Santa Ana, CA) or 0.4 μM aphidicolin (Fisher Bioreagents). Where indicated, inhibitors were used at the following concentrations: AurBi (ZM447439; Biotechne, Minneapolis, MN), 2 μM; Chk1i (AZD7762; Selleck Chemicals, Houston, TX), 2 μM; ATRi (NU6027; EMD Millipore, Billerica, MA), 10 μM; ATMi (KU55933; Selleck Chemicals), 10 μM; and Chk2i (Chk2 inhibitor II; Millipore), 10 μM. As a control for the identification of DNA ultrafine bridges, ICRF-159 (Sigma-Aldrich, St. Louis, MO) was used at a concentration of 10 μM (Chan et al., 2007 (link)).
To induce replication stress, cells were treated for 24 h with either 50 μM hydroxyurea (MP Biomedicals, Santa Ana, CA) or 0.4 μM aphidicolin (Fisher Bioreagents). Where indicated, inhibitors were used at the following concentrations: AurBi (ZM447439; Biotechne, Minneapolis, MN), 2 μM; Chk1i (AZD7762; Selleck Chemicals, Houston, TX), 2 μM; ATRi (NU6027; EMD Millipore, Billerica, MA), 10 μM; ATMi (KU55933; Selleck Chemicals), 10 μM; and Chk2i (Chk2 inhibitor II; Millipore), 10 μM. As a control for the identification of DNA ultrafine bridges, ICRF-159 (Sigma-Aldrich, St. Louis, MO) was used at a concentration of 10 μM (Chan et al., 2007 (link)).