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. 2018 Aug 6;9:3103. doi: 10.1038/s41467-018-05581-y

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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. 6 — Homotrimerization of HIRA is critical for its enrichment at UV damaged sites and for new H3.3 deposition. a (Left, top) Scheme of the experimental assay for HIRA-YFP enrichment at local UV damage in U2OS cells. (Left, bottom) The graph shows the percentage of U2OS cells with HIRA-YFP protein colocalizing with XPB foci. Error bars represent SD from three independent experiments. Statistical analysis using a t-test was performed with Prism 7 software (ns: nonsignificant, *p < 0.05, ***p < 0.001). (Right) Images of representative U2OS cells irradiated locally with UV exhibiting an enrichment of HIRA wt or an absence of enrichment of HIRA (W799A–D800A) at one damage site identified by anti-XPB immunofluorescence. Scale bar 5 μm. b (Top, left) Scheme of the experimental assay for new H3.3 deposition by a Quench-Chase-Pulse experiment (QCP) in HeLa H3.3-SNAP-HA HIRA KO cells transfected with HIRA-HA constructs. (Top, right) Western blot analysis showing the protein expression of exogeneous HIRA-HA wt and (W799A–D800A) mutant. (Bottom, left) Images of cells subjected to QCP: representative images for a cell non-transfected or transfected with HIRA-HA (W799A–D800A) mutant and a high TMR positive representative image for a cell transfected with HIRA-HA wt. (Bottom, right). One graph shows the fluorescence intensity ratio TMR/TMR non-transfected. The other graph gives the percentage of cells exhibiting a positive profile for H3.3 deposition. Error bars represent the SD from three independent experiments. Statistical analysis using a t-test was performed with Prism 7 software (ns: nonsignificant, *p < 0.05)