Extended Data Fig. 8: Ki-67 does not mediate chromosome clustering through PP1 recruitment or through H3(S10) dephosphorylation.
From: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly
a, Schematic of genotyping strategy to endogenously mutate Ki-67’s PP1-binding motif RVSF to RASA. A newly generated SacII restriction site generated by CRISPR–Cas9 nickase as depicted was used to detect correctly mutated alleles. b, SacII restriction fragments were detected by gel electrophoresis following the assay depicted in a, showing successful recombination of all three Ki-67 alleles present in HeLa cells for clone 43 and 96. Showing one example of two biological replicates. c, d, Spindle-less mitotic exit in wild-type cells and in homozygous Ki-67(RASA)-mutant cells. Live cells were imaged in the presence of nocodazole; flavopiridol was added (t = 0 min) to induce mitotic exit. Quantification of chromosome convex hull area, normalized to pre-flavopiridol time points (c) and representative examples stained with SiR-Hoechst (d). Z-projection. Lines and shaded areas indicate mean ± s.d. n = 22 cells (wild-type), n = 23 cells (RASA). e, Molecular organization of the Ki-67 RASA mutant on the surface of mitotic chromosomes before and after flavopiridol addition to taxol treated cells. Ki-67(RASA) was tagged by mCherry and eGFP on either protein end, respectively, and expressed in HeLa cells bearing the endogenous RASA mutation in all three copies of Ki-67. Bars represent mean, significance was tested by a two-tailed unpaired t-test (****P = 8.3 × 10−8, ***P = 0.00049). Chromosome numbers: n = 32 (R-Ki-67-G, pre), n = 28 (R-Ki-67-G, post), n = 37 (G-Ki-67-R, pre), n = 34 (G-Ki-67-R, post). f, g, Immunofluorescence of H3(pS10) during spindle-less mitotic exit in wild-type and Ki-67-KO cells. f, Representative examples of wild-type and Ki-67-KO cells before, 10 and 20 min after flavopiridol addition. Single z-slice is shown. g, Quantification of H3(pS10) mean fluorescence intensity before (wild-type n = 38 cells, Ki-67-KO n = 23 cells) and 5 min (wild-type n = 61 cells, Ki-67-KO n = 61 cells), 10 min (wild-type n = 72 cells, Ki-67-KO n = 65 cells) and 20 min (wild-type n = 73 cells, Ki-67-KO n = 55 cells) after mitotic exit induction with flavopiridol in wild-type and Ki-67-KO cells. Values normalized to average of wild-type 5 min time point. Showing combined data of two independent biological replicates. Bars represent mean, significance was tested with a two-tailed Mann–Whitney test (P = 0.72 for pre-flavopiridol time point, P = 0.96 for 5 min time point, P = 0.71 for 10 min time point and P = 0.26 for 20 min time point). Scale bars, 10 μm.
