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A
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 of mitosis (nuclear envelope breakdown (NEBD) to anaphase onset) was quantified (red bars, median; at least 72 cells, pooled from three independent experiments, were analysed per condition; ****P < 0.0001, ns: not significant, Mann–Whitney test).
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B
Representative time‐lapse sequences of mitotic progression in cells in (A). NEBD corresponds to t = 0. White arrowheads indicate cells undergoing division. Scale bar, 10 µm.
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C, D
Quantification of mitotic defects in cells in (A) (mean; n = 3 independent experiments; > 97 cells quantified per condition; *P < 0.05, **P < 0.01, paired t‐test).
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E
HeLa cells transfected with RNF4 siRNA were synchronized in early S phase by double thymidine block. Six hours after release from the block, cells were pulse‐labelled with 5‐azadC for 30 min. Mitotic cells were isolated by shake‐off 8 h later, washed and incubated or not with MPS1 inhibitor (MPS1i) and collected at the indicated times. Cells were then processed for immunoblotting with indicated antibodies.
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F
Clonogenic survival of 5‐azadC‐treated HeLa cells transfected with indicated siRNAs (mean ± SEM; n = 2 independent experiments).
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G
Clonogenic survival of 5‐azadC‐treated WT HAP1 cells and derivative cell lines expressing a truncated form of RNF4 lacking the RING domain (ΔRNF4; Fig
EV1H) (mean ± SEM;
n = 2 independent experiments).
