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. 2019 Oct 23;10:4834. doi: 10.1038/s41467-019-12772-8

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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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Fig. 3 — Spindle assembly checkpoint activity is not abrogated in tetraploid embryos. a and b Quantification of mitosis duration in 8-cell control (without AZ 3146 n = 47 divisions from seven embryos; with AZ 3146 n = 63 divisions from eight embryos), 4-cell binucleated embryos (without AZ 3146 n = 30 divisions from eight embryos; with AZ 3146 n = 54 divisions from 15 embryos) ***P = 0.0009; ****P < 0.0001 (unpaired Kruskal–Wallis test with Dunn’s test for multiple comparisons) a; 16-cell control (without AZ 3146 n = 61 divisions from seven embryos; with AZ 3146 n = 59 divisions from seven embryos) and 8-cell tetraploid embryos (without AZ 3146 n = 50 divisions from nine embryos; with AZ 3146 n = 63 divisions from 12 embryos) ***P = 0.0005; ****P < 0.0001 (unpaired Kruskal–Wallis test with Dunn’s test for multiple comparisons) b. c Representative z projections of MAD2-positive and MAD2-negative kinetochores demonstrating co-localisation of MAD2 (magenta) and CREST (cyan) in positively stained kinetochores and no co-localisation in negatively stained kinetochores. d and e Proportion of MAD2-positive kinetochores in 8-cell control, 4-cell binucleated d, 16-cell control and 8-cell tetraploid embryos e at 10 mins (8-cell control n = 6 blastomeres; 4-cell binucleated n = 6 blastomeres; 16-cell control n = 5 blastomeres; 8-cell tetraploid n = 5 blastomeres), 20 mins (8-cell control n = 5 blastomeres; 4-cell binucleated n = 6 blastomeres; 16-cell control n = 5 blastomeres; 8-cell tetraploid n = 6 blastomeres) and 30 mins (8-cell control n = 4 blastomeres; 4-cell binucleated n = 6 blastomeres; 16-cell control n = 5 blastomeres; 8-cell tetraploid n = 5 blastomeres) after nuclear envelope breakdown (NEBD). Error bars represent SEM. f and g Representative time-lapse images of live 4-cell binucleated f and 8-cell tetraploid embryos g co-expressing H2B:RFP and MAD1:2EGFP. Note that MAD1:2EGFP signal is clearly observed at the kinetochores shortly after nuclear envelope breakdown (NEBD) and is gradually lost following chromosome alignment at the metaphase plate. Scale bars = 10 µm except for figure c, where scale bars = 1 µm. Where box plots are shown, the centre line represents the median, the bounds of box represent the upper and lower quartiles and the whiskers represent minimum and maximum values