Figure EV4. DPCs in uninterrupted duplex DNA do not activate a DNA damage checkpoint (related to Fig 4).

1. HeLa cells were transfected with indicated siRNAs and synchronized in early S phase by double thymidine block. Following release from the block, cells were pulse‐labelled with 5‐azadC in late S phase for 30 min. Cells were then collected at the indicated times after 5‐azadC withdrawal and analysed by flow cytometry. Data are representative of three independent experiments. Proportion of cells with G2/M DNA content is indicated.
2. RPE1 cells were synchronized in early S phase by double thymidine block. Following release from the block, cells were pulse‐labelled or not with 5‐azadC for 30 min in late S phase, incubated with nocodazole, collected at the indicated times and immunoblotted with indicated antibodies.
3. As in (B), except that SUMOi was added to the culture medium 15 min prior to 5‐azadC treatment.
4. As in (B), except that cells were transfected with RNF4 siRNA prior to double thymidine block synchronization.
5. Immunoblot analysis of RPE1 cells exposed to indicated genotoxic agents and collected 1 h later.
6. Immunoblot analysis of HeLa cells exposed to indicated genotoxic agents and collected 1 h later.