Extended Data Figure 3: Internal chromosome organization is not affected by Ki-67 depletion.

a, b, Hoechst-stained chromosomes of control or Ki-67-depleted HeLa cells were segmented by thresholding, subsequently a convex hull was fitted around segmented chromosomes (red line) (a) and the segmented chromatin area and area of a convex hull fit were quantified (b). The calculated chromatin area of Ki-67 depleted cells was similar to control depleted cells, which demonstrated that Ki-67 depletion has no detectable effect on chromosome-internal compaction. Ki-67 depleted cells lacked inter-chromosomal space as the convex hull area was almost identical with the chromatin area (n = 16 for each siRNA, bars indicate mean ± s.d., ****P < 0.0001 by paired Wilcoxon matched-pairs signed rank test). c, d, Live HeLa cell expressing H2B–mCherry/EGFP–Kleisin-γ were imaged 72 h after siKi-67 no. 1 or control siRNA transfection (c) and mean ± s.d. of sister chromosome axis distance were quantified (d) (n = 508 from 3 experiments (control RNAi), n = 380 from 4 experiments (Ki-67 RNAi), n = 398 from 3 experiments (no RNAi)). e, f, Live HeLa cell stably expressing H2B–mCherry/CENP-A–EGFP were imaged ~60 h after siKi-67 no. 1 or control siRNA transfection (e) and mean ± s.d. of interkinetochore distance were quantified (f) (n = 875 from 3 experiments (control RNAi), n = 1,489 from 5 experiments (Ki-67 RNAi), n = 818 from 3 experiments (no RNAi)). g, Centromere elasticity assay. Metaphase HeLa cells stably expressing H2B–mCherry/CENP-A–EGFP were imaged with 10 s time-lapse before and after nocodazole addition. The interkinetochore distance measurement over time in 12 control cells (control siRNA, light grey; untransfected cells, dark grey) and 10 cells transfected with siKi-67 no. 1 (grey lines) demonstrated that kinetochores were under tension in Ki-67 depleted cells. The red line indicates the mean. h, Chromosome spreads of untransfected or Ki-67 depleted HeLa cells appeared similar. Representative examples of 35 chromosome spreads from 2 independent experiments are depicted. i, Nuclei from control or Ki-67 depleted HeLa cells were treated with titrated amounts of MNase and DNA was subjected to 1.5% agarose electrophoresis. No detectable difference was seen in two independent experiments. Scale bars, 10 μm (a, h), 5 μm (c, e).