Figure S1.
Ensheathed chromosomes in DLD-1 cells after targeted missegregation of Y-chromosome. (A) Schematic diagram after Ly et al. (2017), showing how reexpression of a CENP-A mutant (C-H3) in DLD-1 cells where CENP-A is degraded causes selective misalignment of the Y-chromosome. WT and C-H3 lines were further modified to express mCherry-Sec61β. (B) Western blot of lysates from WT or C-H3 DLD-1 cells treated with doxycycline (Dox) and/or indole-3-acetic acid (IAA) as indicated. Upper blot shows anti-CENP-A detection of endogenous CENP-A fused to EYFP-AID tag (66 kD) and expression of untagged CENP-A (either WT or C-H3). Lower blot shows GAPDH loading control. (C) Typical FISH images locating the Y-chromosome in the main nucleus in control cells and in a micronucleus in cells expressing C-H3 CENP-A. Scale bar, 10 µm. (D) Western blot of lysates from stable cell lines expressing mCherry-Sec61β derived from WT (G2) or C-H3 (G11). Detection of Sec61β or mCherry is shown as indicated with actin loading controls. Migration of Sec61β and mCherry-Sec61β is indicated by black and red arrowheads, respectively. Note that the expression of mCherry-Sec61β downregulates endogenous Sec61β. (E) Deconvolved wide-field microscopy images showing an ensheathed chromosome in G11 cells but not in G2 cells treated with Dox/IAA. Scale bars, 10 µm; 2 µm (insets). (F) Spatially averaged view of all kinetochores in the G11 DLD-1 Dox/IAA dataset (see Materials and methods). Small gray points represent kinetochores at the metaphase plate. Colored points represent misaligned chromosomes that were ensheathed (orange) and those that were not (blue). Spindle poles are shown in black. (G) Box plot to show the relative position of each kinetochore relative to the exclusion zone boundary. Ratio of kinetochores within the exclusion zone are <0 and those within the ER are >0 on a log2 scale. Dots represent kinetochore ratios from 50 DLD-1 cells at metaphase. Boxes show IQR, bar represents the median, and whiskers show 9th and 91st percentiles. Source data are available for this figure: SourceData FS1.

Ensheathed chromosomes in DLD-1 cells after targeted missegregation of Y-chromosome. (A) Schematic diagram after Ly et al. (2017), showing how reexpression of a CENP-A mutant (C-H3) in DLD-1 cells where CENP-A is degraded causes selective misalignment of the Y-chromosome. WT and C-H3 lines were further modified to express mCherry-Sec61β. (B) Western blot of lysates from WT or C-H3 DLD-1 cells treated with doxycycline (Dox) and/or indole-3-acetic acid (IAA) as indicated. Upper blot shows anti-CENP-A detection of endogenous CENP-A fused to EYFP-AID tag (66 kD) and expression of untagged CENP-A (either WT or C-H3). Lower blot shows GAPDH loading control. (C) Typical FISH images locating the Y-chromosome in the main nucleus in control cells and in a micronucleus in cells expressing C-H3 CENP-A. Scale bar, 10 µm. (D) Western blot of lysates from stable cell lines expressing mCherry-Sec61β derived from WT (G2) or C-H3 (G11). Detection of Sec61β or mCherry is shown as indicated with actin loading controls. Migration of Sec61β and mCherry-Sec61β is indicated by black and red arrowheads, respectively. Note that the expression of mCherry-Sec61β downregulates endogenous Sec61β. (E) Deconvolved wide-field microscopy images showing an ensheathed chromosome in G11 cells but not in G2 cells treated with Dox/IAA. Scale bars, 10 µm; 2 µm (insets). (F) Spatially averaged view of all kinetochores in the G11 DLD-1 Dox/IAA dataset (see Materials and methods). Small gray points represent kinetochores at the metaphase plate. Colored points represent misaligned chromosomes that were ensheathed (orange) and those that were not (blue). Spindle poles are shown in black. (G) Box plot to show the relative position of each kinetochore relative to the exclusion zone boundary. Ratio of kinetochores within the exclusion zone are <0 and those within the ER are >0 on a log2 scale. Dots represent kinetochore ratios from 50 DLD-1 cells at metaphase. Boxes show IQR, bar represents the median, and whiskers show 9th and 91st percentiles. Source data are available for this figure: SourceData FS1.

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