UHRF1 regulates EG5 interactions with TPX2 to determine EG5 localization on the spindle. (A) UHRF1 was depleted with siRNAs in DU145 cells. EG5 were stained with immunofluorescent antibodies (red) and the spindle was stained with anti-α-tubulin antibody (green). Scale bar, 5 μm. (B–D) B and C: Corresponding EG5 fluorescence intensity profiles of cells. n = 40 cells for each condition. EG5 fluorescence intensity of the spindle poles (B) or half-spindle (C) was measured, and the percentage of cells at the metaphases with abnormal distribution of EG5 was assessed. n > 50 cells (D). (E) DU145 or PC3 cells were transiently transfected with UHRF1 siRNAs, EG5 protein was immunoprecipitated, and the interacting TPX2 was assessed by immunoblotting. (F) DU145 or PC3 cells were transiently cotransfected with plasmids expressing EG5WT-Flag or EG5K1034R-Flag, EG5 protein was immunoprecipitated with anti-Flag antibody, and the interacting TPX2 was assessed by immunoblotting. (G) DU145 cells with EG5 depletion were transiently transfected with plasmids expressing EG5WT or EG5K1034R. EG5 were stained with immunofluorescent antibodies (red) and the spindle was stained with anti-α-tubulin antibody (green). Scale bar, 5 μm. (H–J) H and I: Corresponding EG5 fluorescence intensity profiles of cells. n = 40 cells for each condition. EG5 fluorescence intensity of the spindle poles (H) or half-spindle (I) was measured, and the percentage of cells at the metaphases with abnormal distribution of EG5 was assessed. n > 50 cells (J). (K and L) DU145 cells with UHRF1 depletion were transiently transfected with plasmids expressing EG5WT-Flag (K), or DU145 cells were transiently transfected with plasmids expressing Flag-tagged wild-type EG5 (WT)/K1034R mutant (L), and immunoprecipitated with a Flag antibody. Half of the immunoprecipitate was used for ATPase assay (bar graph) and the other half was separated by SDS-PAGE and immunoblotted with Flag antibody (gel image). NT, non-transfected. The data for quantification in B–D and H–L are from n = 3 independent experiments. Results are represented as mean ± SD (one-way ANOVA test); error bars represent SD. *, P < 0.05; ***, P < 0.001. Source data are available for this figure: SourceData F5.
Figure 5.

UHRF1 regulates EG5 interactions with TPX2 to determine EG5 localization on the spindle. (A) UHRF1 was depleted with siRNAs in DU145 cells. EG5 were stained with immunofluorescent antibodies (red) and the spindle was stained with anti-α-tubulin antibody (green). Scale bar, 5 μm. (B–D) B and C: Corresponding EG5 fluorescence intensity profiles of cells. n = 40 cells for each condition. EG5 fluorescence intensity of the spindle poles (B) or half-spindle (C) was measured, and the percentage of cells at the metaphases with abnormal distribution of EG5 was assessed. n > 50 cells (D). (E) DU145 or PC3 cells were transiently transfected with UHRF1 siRNAs, EG5 protein was immunoprecipitated, and the interacting TPX2 was assessed by immunoblotting. (F) DU145 or PC3 cells were transiently cotransfected with plasmids expressing EG5WT-Flag or EG5K1034R-Flag, EG5 protein was immunoprecipitated with anti-Flag antibody, and the interacting TPX2 was assessed by immunoblotting. (G) DU145 cells with EG5 depletion were transiently transfected with plasmids expressing EG5WT or EG5K1034R. EG5 were stained with immunofluorescent antibodies (red) and the spindle was stained with anti-α-tubulin antibody (green). Scale bar, 5 μm. (H–J) H and I: Corresponding EG5 fluorescence intensity profiles of cells. n = 40 cells for each condition. EG5 fluorescence intensity of the spindle poles (H) or half-spindle (I) was measured, and the percentage of cells at the metaphases with abnormal distribution of EG5 was assessed. n > 50 cells (J). (K and L) DU145 cells with UHRF1 depletion were transiently transfected with plasmids expressing EG5WT-Flag (K), or DU145 cells were transiently transfected with plasmids expressing Flag-tagged wild-type EG5 (WT)/K1034R mutant (L), and immunoprecipitated with a Flag antibody. Half of the immunoprecipitate was used for ATPase assay (bar graph) and the other half was separated by SDS-PAGE and immunoblotted with Flag antibody (gel image). NT, non-transfected. The data for quantification in B–D and H–L are from n = 3 independent experiments. Results are represented as mean ± SD (one-way ANOVA test); error bars represent SD. *, P < 0.05; ***, P < 0.001. Source data are available for this figure: SourceData F5.

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