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. 2021 Apr 27;11:9016. doi: 10.1038/s41598-021-88512-0

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Nek2 overexpression causes centrosome amplification and binucleation. (a) Representative immunoblot of MCF10A cells expressing either GFP or GFP-Nek2 indicating overexpression of endogenous Nek2 and GFP-Nek2 detected with Nek2 antibodies. (b) CCK8 cell growth/viability assay comparing MCF10A expressing GFP or GFP-Nek2. (c) High-resolution microscopy of a centrosome pair indicating localization of pericentrin (red) and GFP-Nek2 (green). (d) Immunofluorescent staining of pericentrin (red) in GFP and GFP-Nek2-expressing cells (green) counterstained with DAPI (blue). The right inset (GFP-Nek2) indicates a cell with 3 centrosomes. (e) Quantifications of fluorescent microscopy for centrosome amplification by pericentrin staining. N = 3, bars = mean ± SD, *p < 0.05. (f) Immunofluorescent staining of α-tubulin (red) in GFP and GFP-Nek2-expressing cells counterstained with DAPI (blue). The arrow indicates a binucleated cell. (g) Quantifications of fluorescent microscopy for binucleation by α-tubulin staining. N = 3, bars = mean ± SD, *p < 0.05. (h) Representative screenshots of MCF10A/GFP-Nek2 cells subjected to live imaging for 72 h. Centrosome-localized GFP-Nek2 is visible in green and the microtubules are marked by RFP-α-tubulin. Arrows indicate tripolar cell division at different time points.