Fig. 2

Ki-67 degradation leads to collapse of the replication machinery. A Western blot of HCT116:Ki-67-AID cell line blocked with either thymidine or palbociclib 18 h and then treated with or without auxin for 4 h. The blots were probed with antibodies against Ki-67 (first panel), alpha-tubulin (second panel), p21 (third and fourth panels) and γH2AX (fifth panel). B Scheme of how the replisome sequentially assembles and activates. C Representative Western blot of cell fractions (WCL = whole cell lysate, nuclei, and chromatin) of HCT116:Ki-67-AID cell line blocked with thymidine for 18 h and then treated with or without auxin for 4 h. The blots were probed with antibodies against PCNA (first panel), MCM3 (second panel), histone H3 (third and fifth panels) and ORC1 (fourth panel). The images were acquired with a LICOR instrument in the linear range for quantification purposes. The graphic symbol on the right represents the different proteins depicted in the scheme in (B). D Quantification of the blot in (C). The values represent the average of 3 independent replicas and the error bars the standard deviations. The experiments were analysed by Student’s t-test. **p < 0.01, ***p < 0.001. E Representative Western blot of WCL of the HCT116:Ki-67-AID cell line blocked with thymidine 18 h and then treated with or without auxin for 4 h. The blots were probed with antibodies against Ki-67 (first panel), alpha-tubulin (second panel), H3K20me1 (third panel), H3K9me3 (fourth panel), H3k9me2 (fifth panel) and H3k27me2/3 (sixth panel). The images were acquired with a LICOR instrument in the linear range for quantification purposes. F Quantification of the blot in (E). The values represent the average of 3 independent replicas and the error bars the standard deviations. The experiments were analysed by Student’s t-test and did not display any statistically significant changes (ns)