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. 2019 Jan 17;10:273. doi: 10.1038/s41467-018-08043-7

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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 — LUBAC regulates KT-MT attachment, chromosome congression and alignment. a Cold-stable microtubules at pole-proximal kinetochores in control, HOIP or CENP-E depleted mitotic cells. Green, α-Tubulin; red, CREST; blue, DNA. Yellow circles indicate the pole-proximal region (an area covered by a circle with 2.5 μm in radius centered around the spindle pole). Insets show mono-oriented or unattached pole-proximal kinetochores in each group. b Percentage of mono-oriented or unattached pole-proximal kinetochores in control (n = 29 cells), HOIP (n = 25 cells) or CENP-E (n = 25 cells) depleted cells. c Model of LUBAC-dependent CENP-E localization at attached kinetochores on pole-proximal chromosomes regulating KT-MT attachment and chromosome congression. d Cold-stable microtubules on aligned chromosomes in control, HOIP or CENP-E depleted mitotic cells. Green, α-Tubulin; red, CREST; blue, DNA. White dashed boxes indicate areas where α-tubulin intensity was measured. e Relative intensity of cold-stable k-fibers of the attached kinetochores on aligned chromosomes in control, HOIP or CENP-E depleted cells in d. n = 15 cells per group. The average α-tubulin/CREST ratio of each group was normalized to control cells. f CENP-E kinetochore localization in HeLa cells treated with 100 nM proteasome inhibitor Velcade for 2 h, and then released into medium containing DMSO or 1.2 μM Gliotoxin (LUBAC inhibitor) for 30 min. Green, CENP-E; red, CREST; blue, DNA. Dashed boxes, regions of aligned chromosomes. Data are presented as mean ± s.d. of three independent experiments (b and e). Dashed boxes, regions of aligned chromosomes (d and f). ***P < 0.001; two-sided Student’s t-test. Scale bars, 5 μm (main image) and 0.3 μm (magnified region)