Supplementary Figure 2: Irregular chromosome III segregation in Δcen3-NC cells.

(a) Quantitative ChIP enrichment of Cnp1 protein across the Δcen3-NC^ssp2 region in cd3-385 and the Δcen3-NC^rDNA region in cd3-389. The positions of the quantitative PCR probes (a–j) in chromosomes III are shown in Supplementary Figure 1. The ChIP enrichments were normalized to those of the cnt2 region in cen2 (cen2). The data are represented as the average + s.e.m. of n = 3 biological repeats. (b) A typical fluorescent microscopy image of GFP-Cnp1-visualized centromeres (green), Nuc1-mCherry-visualized rDNAs⁵⁰ (magenta), and Hoechst 33342-stained DNAs (blue) of Δcen3-NC cells (cd3-385) harboring the nda3-KM311 cold sensitive mutation³⁵. The cells were arrested in mitosis by incubation at 18°C for 10 h. Cells exhibiting more than two GFP-Cnp1 dots connected to an rDNA signal, in addition to two other GFP-Cnp1 dots corresponding to canonical cen1 and cen2, are indicated by arrowheads. The cell that had lost chromosome III (no rDNA signal) is indicated by an arrow. Scale bar, 10 μm.(c) The percentages of cells exhibiting loss or gain of chromosome III. The indicated strains were mitotically arrested as described in b. The rDNA signals tended to be clustered and were difficult to distinguish even in the nda3-arrested nucleus; therefore, the numbers of GFP-Cnp1 dots associated with the rDNA-containing chromosome in each nucleus were counted. Increases in irregular numbers of rDNA-connected GFP-Cnp1 dots (colored bars) in Δcen3-NC^rDNA cells (cd3-389) and Δcen3-NC^ssp2 cells (cd3-385) are indicative of chromosome III mis-segregation. 7.4% of the Δcen3-NC^ssp2 cells harbored an rDNA-containing chromosome III without any GFP-Cnp1 dots (cyan), suggesting a loss of NC from the chromosome. Note that the quantification of GFP-Cnp1 signal intensities shown in Figure 2d was performed using only the cells exhibiting a regular single GFP-Cnp1 dot connected to an rDNA signal (gray).