Outline of experimental set up to monitor DNMT1 DPC levels in S phase and mitotic cells.
HeLa cells released from double thymidine block in early S phase were mock‐treated or pulse‐labelled for 30 min with 5‐azadC in the presence of SUMOi in late S phase. Following 5‐azadC removal, cells were incubated with SUMOi and nocodazole, and mitotic cells were collected by shake‐off, as outlined in (A). Soluble and chromatin‐enriched fractions were immunoblotted with indicated antibodies.
U2OS cells transfected with indicated siRNAs and then treated as in (B) were collected in late S phase or mitosis (M), subjected to stringent pre‐extraction and immunostained with DNMT1 antibody. DNMT1 foci formation was quantified by QIBC analysis (red bars, mean; > 7,400 cells analysed per condition). Data are representative of three independent experiments.
HeLa cells transfected with indicated siRNAs and synchronized in early S phase by double thymidine block were pulse‐labelled with 5‐azadC in late S phase as outlined in (A). 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.
HeLa cells transfected with indicated siRNAs were treated with 5‐azadC and/or SUMOi in late S phase as outlined in (A). 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.
HeLa cells transfected with indicated siRNAs were pulse‐labelled or not with 5‐azadC for 30 min in late S phase according to the experimental setup in (A). Following 5‐azadC withdrawal cells were incubated with nocodazole, collected at the indicated times and immunoblotted with indicated antibodies.
HeLa cells transfected or not with indicated siRNAs were synchronized in early S phase by double thymidine block, released and pulse‐labelled 5,5 h later with 5‐azadC for 30 min in the presence or absence of SUMOi. Following 5‐azadC removal, cells were subjected to live‐cell imaging analysis, and the duration from late S phase to mitotic entry (nuclear envelope breakdown (NEBD)) was quantified (red bars, median; at least 83 cells, pooled from three independent experiments, were analysed per condition; **P < 0.01, ns: not significant, Mann–Whitney test).
Immunoblot analysis of siRNA‐treated HeLa cells that were exposed to indicated genotoxic agents and collected 1 h later.
