Fig. 3

Ki-67 degradation activates the interferon response pathway. A Volcano plot of the differentially expressed genes obtained by RNA-seq of the HCT116:Ki-67-AID cell line blocked with thymidine for 18 h and then treated with or without auxin for 4 h. In red are indicated genes that belong to the interferon response. The pink line represents p-value < 10e⁻²⁰, the hot pink line p-value = 10e⁻²⁰–10e⁻³⁰ and the purple line p-value = 10e⁻²⁰–10e⁻⁶⁰. B STRING analyses of the upregulated genes. The numbers next to the categories represent the false discovery rate. C HCT116:Ki-67-AID cells were transfected with a plasmid carrying the luciferase gene under the control of the Interferon β promoter together with a plasmid carrying Renilla. Cells were treated with thymidine for 18 h, then with auxin for 4 h, and released in RO3306 for 18 h. The graph represents the luciferase activation normalised to Renilla in cells untreated (Ki-67-AID) or treated with auxin for 4 h (Ki-67-AID auxin). The values represent the average of 3 independent experiments, and the error bars are the standard deviations. The experiments were analysed by Student’s t-test. **p < 0.01. D Scheme for the transfection set-up used for the experiments in (E). E Quantification of the luciferase essays performed as in (D) at 72 h post-transfection for the different cell lines. Oligo 1 and oligo 2 are two independent oligos against Ki-67. The values represent the average of 3 independent replicas, and the error bars are the standard deviations. The experiments were analysed by Student’s t-test. ***p < 0.001. F Representative images of the comet essay of the HCT116:Ki-67-AID cell line blocked with thymidine 18 h and then treated with or without auxin for 4 h. G Quantification of the comet length. The violin plots represent the distribution of the comet tail length in μm. The box inside the violin represents the 75th and 25th percentile, whiskers are the upper and lower adjacent values and the line is the median. A Wilcoxon test was conducted for comparing the experiments and ***p < 0.001. Sample size: control = 234, auxin = 225. H Scheme of the experiment in (I) and (J). I The graph represents the percentage of EdU positive cells for HCT116:Ki-67-AID wt without (−) and with (+) auxin and HCT116:Ki-67-AID STING KO without (−) and with (+) auxin. The values are the average of 2 biological replicas, and the error bars represent the standard deviations. Samples size: HCT116:Ki-67-AID (−)auxin = 224, (+)auxin = 243, HCT116:Ki-67-AID STING KO (−)auxin = 290, (+)auxin = 236. The data were statistically analysed with a chi-squared test. **p < 0.01, ***p < 0.001. J Distribution of the replicating cells according to the patterns shown in (Fig. 1G) from the experiment in (H–I). The data were statistically analysed with a chi-squared test. ***p < 0.001, ns = not significant