Inhibition of Plk1 prevents disengaged daughter centrioles from acquiring PCM but does not suppress precocious centriole disengagement. (A) Precocious centriole disengagement caused by G₂/M phase arrest was suppressed by BI2536 (Plk1 inhibitor; 100 nM). HeLa cells were synchronized in the G₁/S phase by aphidicolin (1.2 µg/ml) for 17 h, then released into fresh medium for 4 h. Next, the cells were treated with DMSO (control), RO3306 (Cdk1 inhibitor; 10 µM), or both of RO3306 and BI2536 (100 nM) for 24 h. HeLa cells were immunostained with antibodies against centrin (green) and Cep192 (red). White arrowheads indicate centrioles. Scale bar, 5 µm. (B) Histograms represent the frequency of the interphase cells with the indicated phenotype observed in A. Values are mean percentages ± SD from three experiments (n = 50 for each experiment). (C) Precocious centriole disengagement in Cep57/Cep57L1-depleted cells was not suppressed by BI2536. HeLa cells were treated with siControl or siCep57/Cep57L1 for 24 h, followed by treatment of DMSO (control) or BI2536 (100 nM) for an additional 24 h. The cells were immunostained with antibodies against centrin (green) and CENP-F (red). White arrowheads indicate centrioles. Scale bar, 5 µm. (D) Histograms represent the frequency of cells in the G₂ phase with the indicated phenotype observed in C. Values are mean percentages ± SD from three experiments (n = 30 for each experiment). (E) BI2536 suppressed recruitment of PCNT at disengaged daughter centrioles. HeLa-GFP-centrin1 cells were treated as in C and immunostained with antibodies against GFP (green), CENP-F (red), and PCNT (cyan). White and black arrowheads indicate PCNT-positive and PCNT-negative centrioles, respectively. Scale bars, 5 µm. (F and G) Histograms represent the frequency of cells with more than two PCNT or γ-tubulin foci among the G₂ phase cells with disengaged or more than four centrioles. Values are mean percentages ± SD from three experiments (n = 30 for each experiment). (H) Schematic illustration of the results in Fig. 4, C–G. The activity of Plk1 is required for the recruitment of PCM components at the disengaged daughter centrioles, but not for precocious centriole disengagement itself. M and D indicate mother and daughter centrioles, respectively. Tukey’s multiple comparisons test was used in D against the total value of centriole disengagement and more than four centrioles to obtain the P value. A two-tailed, unpaired Student’s t test was used in F and G to obtain P values. **, P < 0.01; *, P < 0.05; NS, P > 0.05.