Aurora A phosphorylates KIF4A on off-axis chromosomes. (A) At 7 h after thymidine release, HeLa cells were treated with 300 nM CENP-E inhibitor (GSK923295) for 3 h to accumulate chromosomes at the spindle poles. The cells were then stained for DNA, Aurora A, and Aurora B. (B) CRISPR-edited EGFP-KIF4A cells transfected with either siControl or siKIF4A for 48 h and treated with CENP-E inhibitor for 1 h were stained for KIF4A pT799 and DNA. (C) Purified KIF4A was treated with Aurora A in the presence or absence of Aurora A or B inhibitors. The samples were Western blotted for total KIF4A and pT799. (D and E) KIF4A pT799 staining compared with KIF4A (D) and CENP-A (E) following 10 min treatment with DMSO (control), Aurora A inhibitor (Aur A-i; 0.5 µM MLN8237), or Aurora B inhibitor (Aur B-i; 0.2 µM ZM447439). Arrows A and B mark the phosphorylation signals of KIF4A on chromosomes in the Aurora A and B regions of the spindle, respectively. The enlargements on the right show the pT799 signal in the same regions. (F) The graph shows the intensity of pT799 signal in control (n = 10), Aurora A inhibited (n = 11), and Aurora B inhibited (n = 10) cells at the spindle poles and on the metaphase plate. Error bars and SD are shown. (G) Western blots show the specificity of the Aurora A and B inhibitors in cells under these conditions.