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. 2019 Nov 4;9:15930. doi: 10.1038/s41598-019-50702-2

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Mutations ablating the Tyr pocket of the PLK1 PBD perturb the dynamics of its intracellular localisation during the cell cycle. (A) Representative maximal-intensity projections of kinetochores (KT) in red, centrosomes (CENT) in white, GFP-PLK1_Wt/AAD/AM in green and DNA in blue used for quantification of GFP-PLK1 intensity in (B,C). Cells were treated with Dox (0.5 mg.ml⁻¹) for 7 h, fixed and stained for kinetochores with CREST antiserum, centrosomes with anti-Pericentrin and DNA with Hoechst 33342 and analysed by immunofluorescence microscopy for GFP signal in prophase mitotic cells. Segmentation images show computationally generated segmentations of the DNA (blue), KTs (red) and CENTs (white) used for analysis. Scale bar is 5μm on top panel and 1μm in bottom panel. The images were smoothed, and brightness and contrast settings are constant throughout. (B) Quantification of intensity ratios of GFP-PLK1_Wt/AAD/AM on CREST-stained kinetochores (KT) normalized to the corresponding GFP-PLK1 expression in cells. Data from each cell is represented as a hollow circle, horizontal line (red) indicates mean intensity ratio and error bars indicate ± S.D. Statistical analysis was done using non-parametric, Mann-Whitney two-tailed test with 95% confidence interval, ***p < 0.0001, ns = not significant. (C) Quantification of intensity ratios of GFP-PLK1_Wt/AAD/AM on Pericentrin-stained Centrosomes (CENT) normalized to the corresponding GFP-PLK1 expression in cells. Data from each cell is represented as a hollow circle, horizontal line (red) indicates mean intensity ratio and error bars indicate ± S.D. Statistical analysis was done using non-parametric, Mann-Whitney two-tailed test with 95% confidence interval, ns = not significant.