Figure 3.
Figure 3. The SAH domain is required for the SAC and kinetochore phosphorylation in human cells. (A) DoM for HeLa cells with taxol, pretreated with control siRNA or INCENP siRNA and complemented with hINCENP FL or hINCENP-ΔSAH (ΔSAH). Data are displayed as a kernel density estimate of DoM for all cells in each condition. A box plot with a circle at the median and whiskers extending to ±1.5 × IQR is under each density, n values indicated under sample name, siRNA treatment indicated by gray (siControl) or black (siINCENP) circles adjacent to the sample name. (B) Same as A, except cells were treated with monastrol. (C) Normalized DoM for all cells (gray) and for cells undergoing death in mitosis (red) or mitotic slippage (blue). Data aggregated from at least five independent experiments. All values were normalized to the median of hINCENP in that experiment. Maximum height of each curve is proportional to percentage composition of that class in the aggregate. Red numbers on the right indicate the overall percentage of cells dying in mitosis. The difference in the normalized DoM between cells complemented with FL or ΔSAH hINCENP is also indicated. (D and E) Immunofluorescence quantification of SAC proteins (D) and Aurora B–dependent phosphorylation (E) in taxol-treated cells. In D, the data represent integrated intensity at the kinetochore from n ≥ 18 cells for Mad1 and n ≥ 86 cells aggregated from three independent experiments for Bub1 and BubR1. In E, the data represent integrated intensity at the centromere for n ≥ 52 cells aggregated from two experiments for CENP-A S7ph, integrated intensity on chromatin for n ≥ 20 cells for H3S10ph, and n ≥ 1,800 individual kinetochore measurements for Dsn1 S100ph and Knl1 S24ph using a marker epitope. Representative images approximating the median are presented. Median, IQR (box), and ±1.5 × IQR (error bars) are shown. Two-tailed Mann–Whitney t test; ns, not significant; **, P ≤ 0.01; ***, P ≤ 0.001. Bars, 2 µm. (F) Immunofluorescence quantification of Hec1 S44ph at individual kinetochores standardized to total Hec1 at that kinetochore. Data are visualized as a kernel density estimate, n values indicated under the sample name. ZM, Aurora B inhibitor ZM447439. The maximum height of the density for ZM-treated cells was scaled independently.

The SAH domain is required for the SAC and kinetochore phosphorylation in human cells. (A) DoM for HeLa cells with taxol, pretreated with control siRNA or INCENP siRNA and complemented with hINCENP FL or hINCENP-ΔSAH (ΔSAH). Data are displayed as a kernel density estimate of DoM for all cells in each condition. A box plot with a circle at the median and whiskers extending to ±1.5 × IQR is under each density, n values indicated under sample name, siRNA treatment indicated by gray (siControl) or black (siINCENP) circles adjacent to the sample name. (B) Same as A, except cells were treated with monastrol. (C) Normalized DoM for all cells (gray) and for cells undergoing death in mitosis (red) or mitotic slippage (blue). Data aggregated from at least five independent experiments. All values were normalized to the median of hINCENP in that experiment. Maximum height of each curve is proportional to percentage composition of that class in the aggregate. Red numbers on the right indicate the overall percentage of cells dying in mitosis. The difference in the normalized DoM between cells complemented with FL or ΔSAH hINCENP is also indicated. (D and E) Immunofluorescence quantification of SAC proteins (D) and Aurora B–dependent phosphorylation (E) in taxol-treated cells. In D, the data represent integrated intensity at the kinetochore from n ≥ 18 cells for Mad1 and n ≥ 86 cells aggregated from three independent experiments for Bub1 and BubR1. In E, the data represent integrated intensity at the centromere for n ≥ 52 cells aggregated from two experiments for CENP-A S7ph, integrated intensity on chromatin for n ≥ 20 cells for H3S10ph, and n ≥ 1,800 individual kinetochore measurements for Dsn1 S100ph and Knl1 S24ph using a marker epitope. Representative images approximating the median are presented. Median, IQR (box), and ±1.5 × IQR (error bars) are shown. Two-tailed Mann–Whitney t test; ns, not significant; **, P ≤ 0.01; ***, P ≤ 0.001. Bars, 2 µm. (F) Immunofluorescence quantification of Hec1 S44ph at individual kinetochores standardized to total Hec1 at that kinetochore. Data are visualized as a kernel density estimate, n values indicated under the sample name. ZM, Aurora B inhibitor ZM447439. The maximum height of the density for ZM-treated cells was scaled independently.

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