KBP overexpression alters the localization of both KIF18A and KIF15 on the mitotic spindle. (A) Localization of endogenous KIF18A in metaphase cells arrested in MG132 after the indicated treatments (OE, overexpression). HEC1 was used as a kinetochore marker. (B) Line scans measuring KIF18A distribution along kinetochore MTs. Multiple line scans were averaged after normalizing to peak HEC1 fluorescence intensity. KIF18A, green; HEC1, red; α-tubulin, blue. Solid line indicates the mean, and error bars represent SD. Bottom: Overlay of average KIF18A localization for all three conditions: control siRNA, black; KBP siRNA, blue-green; and mCherry-tagged KBP (mCh-KBP), red. ns, not significant; ****, P < 0.0001 by F-test comparing slopes of linear regressions. (C) Localization of endogenous KIF15 in metaphase cells under the indicated treatment conditions. (D) Quantification of KIF15 localization on the spindle in cells overexpressing KBP. The FWHM of KIF15 intensity was determined for each cell analyzed. ns, not significant; ****, adjusted P < 0.0001 with 95% confidence interval by one-way ANOVA with Tukey’s multiple comparisons test. Error bars represent SD. (E) Correlation plot of KIF15 FWHM alignment values (right y axis, purple) and slopes of KIF18A accumulation (left y axis, red) as a function of mCh-KBP fluorescence in individual cells. KIF18A distribution slopes were averaged from two to four line scans per cell. Black dot denotes average KIF18A accumulation slope under control siRNA condition with standard deviation. Dotted lines are a linear regression showing the data trend. Data in B and D obtained from three independent experiments each with the following cell and line scan numbers: (B) control siRNA (21 [61 lines]), KBP siRNA (22 [63 lines]), mCh-KBP OE (19 [53 lines]); (D) control siRNA (51), KBP siRNA (59), and mCh-KBP OE (24).