The N-terminal region of KNL1 is sufficient to recover Aurora B activity at kinetochores. (A) Quantification of kinetochore fluorescence intensities measured using the Hec1 9G3 antibody in stable HeLa FlpIn T-Rex cell lines expressing KNL1 fragments; n ≥ 100 kinetochores and n ≥ 5 cells. No statistical differences were found between control and HeLa stable cell lines expressing KNL1 fragments. (B) Quantification of phospho-Dsn1 at kinetochores in cells expressing KNL1 fragments; n ≥ 100 kinetochores and n ≥ 5 cells, n = 3 independent experiments. For control and KNL1 siRNA panels, cells were stained with ACA and pDsn1 antibodies. For doxycycline-induced cell lines, cells were stained with pDsn1 antibodies and the GFP fluorescence is shown. The 300N cell line is shown in the panel for control and KNL1 siRNA. (C) Quantification of phospho-INCENP at kinetochores in cells expressing KNL1 fragments. The data shown are from a single representative experiment out of two repeats. For the experiment shown, n ≥ 100 kinetochores and n ≥ 5 cells. The 300N cell line is shown in the panel for control and KNL1 siRNA. Error bars represent SD between cells (Mann-Whitney rank sum test). (A and B) In all cases error bars represent SD between independent experiments. ***, P < 0.001; NS, not statistically significantly different (Mann-Whitney rank sum test). (D) Ratios between the average Aurora B pT232 and pINCENP fluorescence intensities and between Aurora B pT232 and AIM1 fluorescence intensities for each KNL1 mutant cell line are shown as bar graphs. The pT232/pINCENP ratio suggests that pT232 and pINCENP levels are highly correlated between cell lines. The pT232/cenABK(AIM1) ratio suggests that pT232 and cenABK are not highly correlated. See Fig. 3, C and G, and panel C above for n values and statistics. Bars, 5 µm.