The C terminus of Kif18b contributes to Kif18b targeting to microtubule plus ends and the control of microtubule length in vivo. (A) Representative immunofluorescence images of Kif18b-KO HeLa cells transfected with Kif18b-GFP constructs and stained with an anti-EB1 antibody. (B) Quantification of GFP/EB1 signal intensity ratio at microtubule (MT) plus ends. Each data point represents one cell for which a mean of four comet intensities were measured and averaged (mean ± SD). Kif18b-GFP transfection was repeated twice; all others were repeated three times. (C) Mean linescan profile of measured EB1 comets and their corresponding linescan profiles for GFP-Kif18b constructs from cells measured in B. (D) Representative immunofluorescence images of Kif18b-KO HeLa cells transfected with Kif18b constructs, stained with Hoechst for DNA, and immunostained with anti-Ndc80 and antitubulin antibodies. Cells were treated with the Aurora kinase A and B inhibitors MLN8237 and ZM447439. (E) Quantification of microtubule length in cells treated with STLC and MLN8237/ZM447439 after transfection of Kif18b-GFP constructs (111 > n > 66). (F) Time-lapse imaging series of Kif18b-KO HeLa cells transfected with Kif18b-GFP and Kif18b_SD-GFP, imaged with SiR-tubulin and CellMask dye. (G) Quantification of the displacement of the spindle from the center of the cell during metaphase (mean ± SD). Each data point represents spindle displacement over 30–40 min. Asterisks indicate ordinary one-way ANOVA significance value: ***, P < 0.001; ****, P < 0.0001. Bars, 10 µm.