PLK1 is crucial for PCM pole formation and bipolar spindle formation in one-centrosome cells. (A–D) PLK1 and phosphorylated PLK1 observed in one-centrosome cells. (A) Red, green, and blue represent PLK1, GT335, and DNA, respectively. Z-projections: 20 planes, 1 µm apart. Scale bar, 10 µm. (B) Frequency of localization of PLK1 in A. Values are presented as mean percentages from three independent experiments (n > 30 cells for each experiment). (C) Red, green, and blue represent phosphorylated PLK1, centrin, and DNA, respectively. Z-projections of 20 sections, every 1 µm. Scale bar, 5 µm. (D) Frequency of localization of phosphorylated PLK1 in C (n = 6, triplicates, two independent experiments, at least 20 cells in each assay). (E) Mitotic spindle structures upon PLK1 inhibition (1 nM of BI 2536) with or without 100 nM of centrinone. HeLa cells expressing EGFP-centrin1 and pericentrin-mCherry were observed with a 63× objective. Green, red, gray, and blue represent GFP (centrin1), RFP (pericentrin), α-tubulin, and DNA, respectively. Z-projections: 10 planes, 0.3 µm apart. Scale bar, 5 µm. (F) Frequency of mitotic spindle structures in E. Values are mean percentages from two independent experiments (n = 50 for each experiment). (G) The signal intensity of RFP (pericentrin) on GFP (centrin) of fixed mitotic HeLa cells expressing EGFP-centrin1 and pericentrin-mCherry (n > 45 for each condition). Line and error bars represent median with interquartile range. Kruskal–Wallis test was used to determine the significance of the difference. *, P < 0.05; **, P < 0.0001.