EG5 localization and its interaction with TPX2 are affected by UHRF1 mutation. (A) HEK293T, DU145, or PC3 cells were transiently transfected with UHRF1 siRNAs, and UHRF1 or EG5 expression was evaluated by Western blotting. (B) DU145 or PC3 cells with UHRF1 depletion were transiently transfected with plasmids expressing UHRF1^ΔRING or UHRF1^WT, EG5 protein was immunoprecipitated, and the interacting TPX2 was assessed by immunoblotting. (C) DU145 cells with UHRF1 depletion were transiently transfected with plasmids expressing UHRF1^ΔRING or UHRF1^WT. EG5 was stained with immunofluorescent antibodies (red), and the spindle was stained with anti-α-tubulin antibody (green). Scale bar, 5 μm. (D and E) Corresponding EG5 fluorescence intensity profiles of cells. n = 40 cells for each condition. EG5 fluorescence intensity of the spindle poles or half-spindle was measured (D), and the percent of cells at the metaphases with abnormal distribution of EG5 was assessed. n > 50 cells (E). (F) DU145 cells with EG5 depletion were transiently transfected with plasmids expressing EG5^WT or EG5^K1034R. TPX2 were stained with immunofluorescent antibodies (yellow), and the spindle was stained with anti-α-tubulin antibody (green). Scale bar, 5 μm. (G) Corresponding TPX2 fluorescence intensity profiles of cells. n = 40 cells for each condition. TPX2 fluorescence intensity of the spindle poles or half-spindle was measured. The data for quantification in D, E, and G are from n = 3 independent experiments. Results are represented as mean ± SD (one-way ANOVA test); error bars represent SD. n.s., not significant; ***, P < 0.001. Source data are available for this figure: SourceData FS3.