Fig. 5: Blocking the timely defined dissociation of PLK1 dimers impedes the G2-M transition.

A (Left) HeLa-PLK1–3xMyc cells were transfected with different Flag-tagged Bora variants, Bora WT, Bora S252E, Bora S252A, Bora S497A/T501A (Bora AA), and the triple mutant Bora S252E/S497A/T501A (Bora EAA). Cells were synchronized by thymidine treatment at the G1-S boundary and released for 8 h to reach the G2 phase. Cell lysates were subjected to immunoblotting for Bora, PLK1, and ß-Actin. (Right) The relative amounts of exogenously expressed Bora variants were quantified. (Middle) Lysates of transfected HeLa-PLK1–3xMyc cells were subjected to IP using anti-Myc and immunoblotted as indicated. B (Left) Bora-depleted HeLa-PLK1–3xMyc cells were rescued with Flag-tagged Bora WT or Bora EAA and synchronized by thymidine arrest to the G1-S boundary. Cell lysates were immunoblotted for Bora and β-Actin. (Middle) HeLa-PLK1–3xMyc rescued with Flag-tagged Bora WT or Bora EAA were synchronized by thymidine treatment at the G1-S boundary and released for the indicated time points. Cell lysates were immunoblotted for PLK1, Bora, Aur-A, Cyclin B1, and β-Actin. (Right) The lysates were subjected to anti-Myc IP and blotted for PLK1 and PLK1-pT210. C Bora-depleted HeLa-PLK1–3xMyc cells expressing mcherry-Histone H2B cells were rescued with Flag-tagged Bora WT or Bora EAA, synchronized by thymidine treatment at G1/S, and released for 16 h. (Left) The mitotic indices have been assessed using time-lapse microscopy. The percentages of mitotic cells over time in each treatment group is represented in the line diagram. The results are presented as means ± SD (n = 150). (Right) Representative Figures of time-lapse analysis. The arrowheads point to mitotic cells.