(
A–D) Quantification of KIF22 fluorescence intensity in untreated HeLa-Kyoto cells transfected with control siRNA (
A), cells treated with doxycycline to induce expression and transfected with control siRNA (
B), untreated cells transfected with KIF22 siRNA (
C), and cells treated with doxycycline and transfected with KIF22 siRNA (
D) normalized to the mean intensity of uninduced, control knockdown cells (endogenous KIF22 expression level) for each cell line (
A). Thirty-two GFP, 25 KIF22-GFP,28 KIF22-GFP T463D, and 31 KIF22-GFP T463A untreated cells transfected with control siRNA (
A), 29 GFP, 27 KIF22-GFP, 27 KIF22-GFP T463D, and 29 KIF22-GFP T463A doxycycline-treated cells transfected with control siRNA (
B), 25 GFP, 26 KIF22-GFP, 23 KIF22-GFP T463D, and 26 KIF22-GFP T463A untreated cells transfected with KIF22 siRNA (
C), 28 GFP, 28 KIF22-GFP, 31 KIF22-GFP T463D, and 26 KIF22-GFP T463A doxycycline-treated cells transfected with KIF22 siRNA (
D), from three experiments. Bars represent means. P values from Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. P values are greater than 0.05 for comparisons without a marked p value. (
E) Plotting background-subtracted GFP intensity against the distance between separating chromosome masses at 7 min indicates that this distance is dependent on expression level (Spearman correlation coefficient –0.3964, one-tailed p-value=0.0004). Gray dashed line indicates mean background subtracted GFP intensity of 100. Thirteen KIF22-GFP, 32 KIF22-GFP T463D, and 24 KIF22-GFP T463A cells from five experiments. (
F) Distance between separating chromosome masses of cells expressing lower levels of KIF22-GFP (mean background subtracted GFP intensity less than 100). Lines represent the mean and the shaded area denotes SEM. Seven KIF22-GFP cells from four experiments, 17 KIF22-GFP T463D cells from five experiments, and 14 KIF22-GFP T463A cells from four experiments. (
G) Distance between separating chromosome masses 7 min after anaphase onset of cells expressing lower levels of KIF22-GFP (mean background subtracted GFP intensity less than 100). Bars indicate medians. P values from Kruskal-Wallis test. P values are greater than 0.05 for comparisons without a marked p value. Seven KIF22-GFP cells from four experiments, 17 KIF22-GFP T463D cells from five experiments, and 14 KIF22-GFP T463A cells from four experiments. (
H) DAPI-stained nuclei of Hela-Kyoto cells. Fixed approximately 24 hr after treatment with doxycycline to induce expression. Scale bar 20 μm. Images are representative of three or more experiments. (
I) Schematic depicting the measurement of chromosome signal intensity in anaphase and the use of the full width at half maximum (FWHM) as a measure of anaphase chromosome mass broadness. (
J) FWHM of the plotted intensities of separating chromosome masses of HeLa-Kyoto cells expressing KIF22-GFP or KIF22-GFP T463A. Lines represent the mean and the shaded area denotes SEM. (
K) Minimum FWHM value, representing maximal anaphase chromosome compaction, between cells expressing KIF22-GFP and KIF22-GFP T463A. P value from Mann-Whitney test. Bars represent medians. Data in (
J) and (
K) represent 12 KIF22-GFP and 24 KIF22-GFP T463A cells (24 KIF22-GFP and 48 KIF22-GFP T463A chromosome masses) from five experiments. See
Figure 8—figure supplement 1—source data 1.