Extended Data Fig. 7: Ki-67 is required for chromosome clustering in Ki-67-knockout cells after histone overexpression or histone hyperacetylation.
From: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly
a, Time-lapse microscopy of clustering-deficient HeLa Ki-67-KO cell stably expressing H2B–mCherry to high levels during a flavopiridol-induced mitotic exit in the presence of nocodazole. Transient expression of eGFP–Ki-67 to levels at least matching Ki-67 endogenous levels (lower panel). Single z-slice shown. b, Chromosome convex hull area quantification, normalized to pre-flavopiridol time points, of 29 Ki-67-KO and 28 Ki-67-KO + eGFP–Ki-67cells as in a. c, Immunoblot analysis of H2B from cell lysates, isolated from wild-type cells (1) or cells transiently expressing H2B–mNeonGreen (2, 3). Sample 3 was lysed after 48 h, whereas sample 2 was additionally FACS sorted for the 10% brightest cells which we estimated to represent the population of cells that suppress the Ki-67-knockout individualization failure phenotype. For gel source data, see Supplementary Fig. 1. n = 2 biological repeats. d, e, Immunofluorescence of acetylated H3 of Ki-67-KO cells arrested in nocodazole with or without trichostatin A. d, Representative examples. Single z-slices shown. e, Quantification of acetylated H3 mean fluorescence intensity in Ki-67-KO cells arrested in nocodazole with and without trichostatin A. Bars represent mean, significance was tested with a two-tailed Kolmogorov–Smirnov test. (****P = 2.22 × 10−16). n = 115 cells (Ki-67-KO), n = 111 cells (Ki-67-KO + TSA). f, Time-lapse microscopy of HeLa wild-type or clustering-deficient Ki-67-KO cells with or without transient re-expression of Ki-67 during a flavopiridol-induced mitotic exit in the presence of nocodazole. Cells were treated with trichostatin A 2 h before imaging to rescue the Ki-67-knockout individualization failure phenotype in Ki-67-KO cells. Representative examples stained with SiR-Hoechst, single z-slices shown. g, Chromosome convex hull area, normalized to pre-flavopiridol time points, quantification of 21 Ki-67-KO, 31 wild-type and 32 Ki-67-KO + eGFP–Ki-67 cells as in f. Lines and shaded areas indicate mean ± s.d. (b, g), scale bars, 10 μm.
