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. 2018 Apr 30;9:1731. doi: 10.1038/s41467-018-04122-x

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© The Author(s) 2018

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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Fig. 1 — CEP85 is a regulator of centriole duplication. a–c Effect of CEP85 depletion on centrosome number. U-2 OS cells expressing Tet-inducible FLAG or the siRNA-resistant FLAG-CEP85 transgene were transfected with control or CEP85 siRNA and induced with tetracycline for 72 h. Scale bar 10 μm, white boxes indicate the magnified region. b The graph shows the percentage of cells with the indicated centrosome numbers (n = 200/experiment, three independent experiments). c Western blot analysis of FLAG-CEP85 protein levels. α-tubulin served as a loading control. d–f Impact of CEP85 depletion on centriole number. The G2-phase arrest assays (see Methods) were performed in U-2 OS cells conditionally expressing FLAG or siRNA-resistant FLAG-CEP85, treated with control or CEP85 siRNA and induced with tetracycline for 72 h. Scale bar 10 μm, white boxes indicate the magnified region. e Bar graph, the number of centrioles per cell were counted. (n = 200/experiment, three independent experiments). f Western blot showing the levels of FLAG-CEP85 in control or CEP85 siRNA transfected cells. α-tubulin served as a loading control. g, h The role of CEP85 in PLK4-induced centriole overduplication. The PLK4 assays were performed as described in Methods. Scale bar 10 μm, white boxes indicate the magnified region. h The graph showing the percentage of cells with over four centrioles (n = 100/experiment, three independent experiments). i, j The role of CEP85 in S-phase arrest induced centriole overduplication. The assays were performed as described in Methods. Scale bar 10 μm, white boxes indicate the magnified region. j The graph indicates the percentage of cells with over four centrioles (n = 100/experiment, three independent experiments). k, l Determining at which stage CEP85 acts in the centriole duplication pathway using the PLK4-induced centriole overduplication assays (see the Methods). Scale bar 10 μm, white boxes indicate the magnified region. l The graph indicates the relative levels of CEP192, Myc-Plk4, STIL, SASS6 and Centrin at centrosomes (n = 100/experiment, three independent experiments). Two-tailed t-test was performed for all p-values, all error bars represent SD, and asterisks for p-values are **p < 0.01 and *p < 0.05