Extended Data Figure 6: Generation of a Ki-67 knockout cell line.

a, CRISPR/Cas9 was used to generate a HeLa cell line with indicated deletions on exon 5 of the Ki-67 allele. b, DNA sequencing chromatogram confirmed that no further alleles are present. c, Metaphase plates of live wild-type HeLa and Ki-67 knockout cells stained with Hoechst (n = 30 per cell line). d, Western blot performed on whole cell lysates of wild-type or Ki-67 knockout cells. The two high molecular weight bands labelled by anti-Ki-67 antibody in wild-type HeLa that corresponded to the two Ki-67 isoforms were undetectable for Ki-67 knockout cells. Actin was used as a loading control. e, Representative time-lapse image series of a Ki-67 knockout cell proceeding from prophase to prometaphase in the presence of nocodazole (n = 12, see Supplementary Video 3). Chromosomes are labelled with SiR-Hoechst and two regions were selected to exemplify coalescence of chromosomes upon their close approach. Arrows mark regions just before their coalescence. f, Automated segmentation of SiR-Hoechst-labelled interphase nuclei of wild-type and Ki-67 knockout cells confirmed a normal nuclear size of Ki-67 knockout cells (mean ± s.d. of 200 wild-type and 270 Ki-67 knockout cells). g, Representative example images of interphase wild-type or Ki-67 knockout cells stained with SiR-Hoechst of the quantification in f. h, The sensitivity of Ki-67 knockout cells to low dose nocodazole, caffeine or a topoisomerase II inhibitor (ICRF-193) was compared to wild-type by a colony formation assay. Representative images from two to three independent experiments are shown. Scale bar, 5 μm (c), 10 μm (e, g).