Figure 5.

Phosphorylation of the kinetochore proteins Hec1 and Dsn1 does not require centromere-concentrated Aurora B. (A–D) IF images of Hec1 S44ph, Aurora B, and CENP-C (A) and Dsn1 S109ph, Aurora B, and CENP-C (C) of HCT116 WT and Haspin CM cells + 10 µM BAY-320, arrested in mitosis using nocodazole from a RO-3306 release (scale bar, 5 µm). (D) IF intensity levels of Hec1 S44ph (B) or Dsn1 S109ph (D) at kinetochores. Levels were normalized over CENP-C. The graphs show the mean and SD. A minimum of 27 cells was quantified per condition. Data are representative of two independent experiments. No significant differences were observed as determined by a two-way ANOVA using Tukey’s multiple comparison test. (E) IF images of mitotic cells depicting DAPI and Lamin B staining. The differences in the morphology of DAPI staining and the decreasing levels of Lamin B allowed us to classify five categories of (early) mitotic cells. Cells were released from RO3036 into ±10 µM BAY-320 for 30 min (scale bar, 5 µm). For each category, we determined the IF intensity levels of Hec1 S44ph (F) or Dsn1 S109ph (G) at kinetochores in HCT116 WT and Haspin CM cells + 10 µM BAY-320.