Fig. 6: Preventing PLK1 dimer dissociation interferes with PLK1 nuclear transport during the G2 phase and causes PLK1 mislocalization.

A Stabilizing PLK1 dimers impedes Aur-A-TPX2 interaction. HeLa-PLK1–3xMyc cells were first transfected with Flag-Bora WT or Flag-Bora EAA. Cells were synchronized by thymidine treatment at the G1-S boundary and released for 10 h to reach mitosis. As a control experiment, similarly transfected but asynchronized cells were used. (Left) The cell lysates were first immunoblotted for the indicated antibodies. (Middle) The lysates were subjected to an anti-Myc IP and blotted for PLK1. (Right) The same lysates underwent an anti-TPX2 co- IP, and the precipitates were stained for TPX2 and Aur-A. (n = 3). B HeLa-PLK1–3xMyc cells were transfected with different Flag-tagged Bora variants, Bora WT, Bora S252E or Bora EAA, followed by a synchronization into the G2 phase using RO3306 (4.5 µM). (Left) Cell lysates were subjected to immunoblotting for Bora, PLK1, and ß-Actin. (Right) Lysates of transfected HeLa-PLK1–3xMyc cells were subjected to IP using anti-Myc or anti-Importin α and immunoblotted for Importin α and PLK1. (n = 3). C Confocal images of HeLa-PLK1–3xMyc. The cells were first transfected with Flag-Bora WT or Flag-Bora EAA. Cells were synchronized by thymidine treatment at the G1/S boundary and released for 10 h to reach mitosis. Next, the cells were fixed and processed for IF using PLK1 and PLK1-pT210 antibodies. The cytoplasmic and DNA-associated PLK1 and the PLK-pT210 signals are shown as representative Figures. The arrowheads show displaced and secluded PLK1 in the cytoplasm. Scale bar: 10 µm (n = 25). D, E The cytoplasmic and DNA-associated PLK1 and PLK1-pT210 intensities in both transfection groups were quantified. The values were calculated from at least 30 cells and represented as a Box-Whiskers plot (****p ≤ 0.001).