Figure S4.

Chromosome Ensemble Geometries in BAF-Depleted Cells, Related to Figure 3

(A–E) Analysis of chromosome ensemble geometries during mitotic exit. (A) Schematic of the measurements shown in (B-E), segmentation was performed by local adaptive thresholding. (B) Height of the chromosome ensemble (curves and range indicate mean ± s.d., complete maximum projection trajectories of chromosome ensembles). Dashed line indicates the time point of initial rebinding of nuclear envelope to the chromosome ensemble (see also Figure 3A) at 7:00 min:s. (C) Chromosome height at 7:00 min:s for data shown in (B; dots indicate individual chromosome ensembles; median with interquartile range is plotted, ns > 0.05 by two-tailed Mann-Whitney test). (D) and (E) width of the chromosome ensembles for the cells shown in (B, C).

(F–H) Distribution analysis of microtubules during mitotic exit. (F) Immunofluorescence staining for Tubulin and Lamin B in HeLa cells 96 hr after siRNA transfection. DNA was stained with Hoechst 33342 (representative images of two independent experiments is shown). Cells were binned into mitotic stages based on chromosome ensemble distance and furrow ingression status. (G) Analysis of cellular microtubule distribution. Chromosomes were segmented by thresholding and the mask (‘chromosomes’) was used to measure tubulin signal within the chromosome ensemble. The mask was then dilated and a band of 1 μm thickness was generated (‘cytoplasm’) to measure cytoplasmic tubulin signal. (H) Quantification of microtubule distribution (median with interquartile range is plotted, ^∗∗∗p < 0.0002, ^∗∗∗∗p < 0.0001 by Kruskal-Wallis with Dunn’s correction for multiple comparison, n ≥ 28 cells per condition from two independent experiments).

(I) Efficacy of 200 ng/ml nocodazole treatment in BAF-depleted and control cells in comparison with prometaphase under normal cell culture conditions.

(J) Long-term imaging of live HeLa cells stably expressing H2B–mCherry and Lap2β–EGFP 72h after siRNA transfection in the presence of 200 ng/ml nocodazole and 320 nM reversine.

(K) Quantification of Lap2β-EGFP fluorescence in internal and rim regions of the chromosome ensemble (see also Figure 3C) 35 min after mitotic exit and normalized to the average signal in the respective control condition (dots indicate chromosome ensembles, lines indicate median and interquartile range, ^∗∗∗∗p < 0.0001, ns > 0.05 by Mann-Whitney test, n ≥ 22 cells from four independent experiments).

Scale bars are 10 μm.