Fig. 4

Ki-67 is in proximity of the replication machinery during S phase. A Scheme of Ki-67-APEX2 tagged protein. B Representative images of the HCT116:Ki-67-APEX2 cell line treated with biotin for 30 min and then with or without H₂O₂ for 1 min. The cells were fixed and stained with RFP-streptavidin and DAPI. Scale bars 5 μm. C Scheme of the SILAC experiment. Drawing made with BioRender. D STRING analyses of the APEX proteome from the experiment in C. E Reactome pathway enrichment analyses of the Ki-67 interacting proteins form the experiment in (C). F Western blot of HCT116 (C) or HCT116:Ki-67-APEX2 (APEX2) probed with antibodies against MCM3 (top panels) and PCNA (bottom panels). G Violin plot of the quantification of the experiment in (H). 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. Sample size: control = 114, auxin = 99. A Wilcoxon test was conducted for comparing the experiments and ***p < 0.001. H Representative images of the proximity ligation assay (PLA) using anti-MCM3 and anti-GFP antibodies on HCT116:Ki-67-AID cell line without (left) of with (right) auxin. Scale bar 5 μm. I Scheme of the experiment for (J) and (K). J Representative images of HCT116:Ki-67-AID cell line at different time points from the thymidine block release. The numbers represent the percentage of cells showing Ki-67 appearing as foci at the nuclear periphery. Cells were stained with DAPI. The yellow arrows indicate the foci. Scale bar 5 μm. K Representative images of HCT116:Ki-67-AID cell line at early (top panels), middle (middle panels) and late (bottom panels) replication stages and stained with anti-BrdU antibodies and DAPI. Scale bar 5 μm. 1 and 2 boxed regions in the left panels represent the enlarged images on the right. The frame colour of the enlargements indicates the specific channel