Fig. 5: Inhibition of RAD51-mediated MiDAS delays anaphase onset in under-replicated cells.

a Schematic diagram of experimental procedure for synchronising HEK293 cells exogenously expressing mCherry-tagged Histone H2B and GFP-tagged Lamin B1. Following double thymidine block and release (dT-B/R) and 5 h RO3306 arrest, cells were released into mitosis (m-R) and mitotic progression was monitored by live microscopy. b Mitotic duration of the parental HEK293 cell line, the RAD51^S14A/- mutant cell line (S14A) and the wild-type RAD51 complemented cell line (S14A + WT) following cell synchronisation as depicted in a. Cells that do not enter into anaphase within 4 h from nuclear envelope breakdown (NEBD) are excluded from analysis. Mitotic duration is measured from NEBD to anaphase onset (AO). Data were obtained from three independent experiments (50 cells analysed per repeat); n = 150 per condition. Data distribution is represented by Tukey box-and-whisker plots. Bounds of box are 25–75th percentile, centre shows the median and ‘+’ marks the mean. Whiskers indicate ±1.5xIQR, data outside this range are drawn as individual dots. p-values were calculated by a Kruskal–Wallis test followed by uncorrected Dunn’s test. Asterisks indicate **p-value ≤ 0.01; ****p-value ≤ 0.0001. c Schematic diagram of experimental procedure for synchronising U2OS cells exogenously expressing mCherry-tagged Histone H2B and GFP-tagged Lamin B1. Following dT-B/R, cells were released into S phase in the absence or presence of 0.4 µM APH ( ±APH-S) followed by 12-h RO3306 arrest. Where indicated, mitotic progression was monitored in the presence of 20 µM RAD51 inhibitor (B02); 2 mM APH (APH-M), 2 µM MPS1 inhibitor (AZ3146), 50 nM neocarzinostatin (NCS) or vehicle (-). All drug treatments were added to cell culture directly after mitotic release (m-R). d Mitotic duration of U2OS cells as measured from NEBD to AO. Cells that do not enter into anaphase within 4 h from NEBD are excluded from analysis. Data was obtained from at least three independent experiments; n indicates the total number of cells monitored per condition. Data distribution is represented by Tukey box-and-whisker plots. Bounds of box are 25–75th percentile, centre shows the median and ‘+’ marks the mean. Whiskers indicate ±1.5xIQR, data outside this range are drawn as individual dots. p-values were calculated by a Kruskal–Wallis test followed by uncorrected Dunn’s test. Asterisks indicate *p-value ≤ 0.05; ***p-value ≤ 0.001; ****p-value ≤ 0.0001. Source data are provided as a Source Data file.