Quantitative analysis of the phosphoregulation of K fibers in metaphase cells. (A) Example metaphase PtK1 cell depleted of endogenous Hec1 and rescued with 1D Hec1, fixed, and stained with antitubulin and anti-Hec1 antibodies. The white box shows the area in which the mean intensity of tubulin staining was measured to compare relative sizes of K fibers in different cells. Bar, 1 µm. (B) Interkinetochore distances in metaphase cells expressing Hec1 with different numbers of phosphomimetic substitutions. Results for 1D Hec1 are averaged between three mutants (Fig. S2 D). Shaded area shows the range for interkinetochore distances in WT Hec1 cells. The broken line shows interkinetochore distance in the absence of MTs: 1.0 µm (DeLuca et al., 2006, 2011). At least 127 kinetochores were analyzed in ≥30 cells for each Hec1 mutant. (C) Relative K-fiber intensities in cells expressing Hec1 with different numbers of phosphomimetic substitutions. Results for 1D Hec1 are averaged between three Hec1 mutants (Fig. S2 E). Mean intensity of K fibers in WT cells was chosen as 1; shaded area shows the corresponding range. The value for 9D Hec1 is significantly different from 0 (P < 0.05), which is predicted from the linear relationship. The black line is a best linear fit of experimental points for zero to four phosphomimetic substitutions. At least five K fibers were measured per cell in ≥22 cells for each Hec1 mutant. Error bars are SEMs. a.u., arbitrary unit.