Kif18b controls astral microtubule length and spindle positioning. (A) Western blot showing depletion and gene KO of Kif18b in HeLa cells using siRNA and CRISPR-Cas9. (B) Representative immunofluorescence images of control and Kif18b-KO HeLa cells acquired using antibodies against Ndc80 and β-tubulin. (C) Representative immunofluorescence images of control and Kif18b-KO HeLa cells treated with Aurora kinase A and B inhibitors MLN8237 and ZM447439 acquired using antibodies against Ndc80 and β-tubulin. (D) Quantification of microtubule length was measured in >30 cells per experiment. Error bars represent SD. (E) Representative time-lapse imaging of cells incubated with SiR-tubulin and CellMask dye after an STLC washout and MG132 treatment. (F) Graph representing the mean spindle length and the corresponding SD for HeLa and Kif18b-KO HeLa cells (n = 32 and 36), defined in E during spindle elongation. Delay in spindle elongation of Kif18b-KO cells was statistically significant. (G) Schematic diagram showing how spindle position, displacement, and rotation are determined. (H) Box-and-whisker plot showing quantification of the spindle displacement from the center of the cell during metaphase. Each point represents the displacement of one spindle over at least 30 min (HeLa, n = 26 cells; Kif18b KO, n = 34 cells). Line in the middle box is plotted as the median, and edges of the box represent 25th and 75th percentiles. The whiskers represent the minimum and maximum displacement of cells. (I) Quantification of the number of cells undergoing >30° rotation in z out of the imaging plane during metaphase (HeLa, n = 86 cells; Kif18b KO, n = 122 cells). Bars, 5 µm. Asterisks indicate unpaired t test significance: ***, P < 0.001; ****, P < 0.0001.