Fig. 2
ATR signalling is the main driver of cell cycle exit in G2 phase. a Schematic representation of the experimental set-up. b, c Mitotic entry was scored in RPE CCNB1YFP-positive (G2)-cells after IR (2 Gy). ATM inhibitor (KU55933) (b) or ATR inhibitor (VE-821) (c) was added at indicated timepoints before or after IR. Average of three independent experiments + sem. d Percentage of G2 cells with nuclear translocated Cyclin B1 at 6 h after OHT addition. S phase cells were excluded based on Edu staining. Prophase cells with nuclear Cyclin B1 localization were excluded based on DNA condensation as stained by DAPI. Average of three independent experiments + sem. e Stills represent G2 cells (Edu negative) 0 h or 4 h after IR (2 Gy) with nuclear translocated or cytoplasmic Cyclin B1 localization. pSer/Thr staining in foci represents ATR substrate phosphorylation. Scale bar 10 μm. f Quantification of total pSer/Thr phosphorylation signal in foci at indicated timepoint after IR in cells with cytoplasmic or nuclear Cyclin B1 localization. Pooled cells from three independent experiments. n > 44. Box plot whiskers indicate 10–90% boundary. ****p < 0.0005 two-sided t-test. g, h Time of mitotic entry after IR (2 Gy) in G2 cells scored in (Fig. 2b, c). Pooled cells from three independent experiments n > 35 + sd. i Stills represent mitotic cells collected by nocodazole and stained for MDC1 and γH2AX to quantify DSBs in cells that entered mitosis within 9 h after IR (2 Gy) in G2 phase. Cells in S phase at the time of IR are excluded by EdU staining. Scale bar 5 μm. j Number of MDC1 and γH2AX double positive foci in mitotic cells as shown in i. Pooled cells from two independent experiments n > 35 + sd
