Human retinal pigment epithelial cells lacking KIF18A function progress through mitosis with unaligned chromosomes. (A) Representative images of centrosomes and kinetochores in fixed hTERT-RPE1 cells treated with the indicated siRNAs. (B) Plot of kinetochore (KT) distribution at the indicated times following siRNA treatment measured using the FWHM of kinetochore fluorescence along the pole-to-pole axis. Data are from two (48 and 96 h) or three independent experiments (144 h). Mean ± SEM is shown. (C) Images of KIF18A KO cells transiently expressing EGFP or EGFP-KIF18A. Cells were fixed and stained for γ-tubulin and DNA. (D) Plot of metaphase plate width in control and KIF18A KO cells expressing GFP or GFP-KIF18A. Plate width was determined by measuring FWHM of DAPI fluorescence along the pole-to-pole axis. Data were collected from two independent experiments. Mean ± SEM is shown. (E) Stills from time-lapse DIC imaging of hTERT-RPE1 cells from the indicated treatment groups. (F) Cumulative frequency plot of time from nuclear envelope breakdown (NEB) to anaphase onset. n = 27 (control), n = 40 (KIF18A KD), and n = 52 (KIF18A KO). Data were from four independent experiments. All statistical comparisons were made using a Kruskal–Wallis one-way ANOVA with Dunn’s multiple comparisons test; *, P < 0.01.