Fig. 5

Ki-67 degradation leads to Huwe1 activation. A STRING analyses of proteins form the APEX2 proteome that are linked to DNA replication (circled in blue). B STRING representation of known HUWE1 interactors. C Scheme of the experiment for (D) and (E). D Quantification of the experiment in (E). The values represent the average of 5 independent biological replicas for the MG132 experiment and 2 independent biological replicas for the BI8626 experiment; the error bars show the standard deviations. The experiments were analysed by chi-squared test. ***p < 0.001. E Representative images of the experiments as indicated in (C). The cells were fixed and stained for H2AX (red) and counterstained with DAPI (blue). Sample size: control = 560, control (MG132) = 620, control (BI8626) = 240, auxin = 578, auxin (MG132) = 531, auxin (BI8626) = 234. Scale bar 5 μm. F–H Representative Western blot analyses of HCT116:Ki-67-AID cell line of the experiment as indicated in C (top). The blots were probed with anti-alpha tubulin or anti-GAPDH antibodies and with anti-H2AX (F), p53 (G) and CHK1 (H). The images were acquired with a LICOR machine in the linear range for quantification purposes. The graphs at the top represent the quantification of the blots. 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 < 0.05 **p < 0.01. I Expression levels of the indicated genes obtained from the RNA seq experiments described in Fig. 3A. The values represent the average of the 3 independent replicas, and the error bars are the standard deviations. The experiments were analysed by Student’s t-test. ns = not significant. J Representative Western blot analyses of HCT116:Ki-67-AID cell line blocked with thymidine for 18 h then untreated (Ki-67-AID) or treated with auxin for 4 h (Ki-67 AID auxin) in the presence (+) or absence (−) of BI8626. The blots were probed with anti-GAPDH and anti-p53 antibodies. The images were acquired with a LICOR instrument in the linear range for quantification purposes. The graph at the top represents the quantification of the blots. 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 test. **p < 0.01 ***p < 0.001