Fig. 5

Fragility, checkpoint activation, and instability induced by quiescence in CIZ1-null cells. A Short SW and AB strategy used to analyse the response of WT (green) and CIZ1-null (blue) nuclei in B and C. B The relative number of nuclei that survived 40 passes through a 25G needle [41] ± SEM, where N represents the number of independent experiments, each with 2–7 fields analysed. C Frequency of cells with activated checkpoint kinase (pATM), ± SEM. Right, representative images showing pATM foci (red) in WT and CIZ1-null AB nuclei. D Checkpoint activation (pATM, pCHK1, yH2AX) in WT and CIZ1-null cells after CI and extended culture, where day 0 represents cycling cells. Below, representative images show foci in contact inhibited CIZ1-null cells at the end of the 21-day time course. E 6 cm culture dishes stained with crystal violet showing primary fibroblasts (tail-tip, TTF and embryonic, PEF) populations from WT and CIZ1-null mice, at the end of the 21-day CI time course. Bar is 1 cm. Right, quantification of macroscopically visible foci in CIZ1-null TTF populations and a PEF population, compared to WT. Histograms show mean foci incidence ± SD at 21 days after plating 4.5 × 10⁵ cells. N represents the number of plates counted. F Field images of WT and CIZ1-null monolayers 21 days post-plating, detected after a 16-h pulse with EdU. Monolayer cells remain unlabelled however CIZ1-null focal outgrowths contain S phase cells. Scale bar 200 μm. G Schematic representation of the order of events observed in CIZ1-null cells following CI and extended culture. Results are compared by t-test (D, E) or two-way ANOVA (C) where ns denotes no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001. DNA is stained with DAPI (blue), and scale bar represents 10 μm unless otherwise stated