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. 2021 Jun 22;12:3835. doi: 10.1038/s41467-021-24153-1

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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 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 — a Fluorescence images showing nascent transcripts labelled with ethynyl-uridine (EU) in unirradiated (0 h) or UVC-irradiated (2 h, 24 h) HeLa cells treated with the indicated siRNAs (siLUC, control). Knock-down efficiencies are controlled by western blot at the time of UV irradiation (Tubulin, loading control). The scheme on the right represents the HIRA complex chaperoning H3.3-H4. The graph shows quantification of nascent transcript levels post UVC relative to unirradiated cells using the same colour code as the scheme for HIRA complex subunits. b, c New H3.3 accumulation at UVC damage sites (marked by CPD immunostaining) analysed 2 h and 24 h post local UVC irradiation in U2OS H3.3-SNAP cells treated with the indicated siRNAs (siLUC, control). The scatter plots show new H3.3 levels at UV sites (mean ± s.d. from at least 86 cells scored in three independent experiments). siRNA efficiencies are controlled by western blot for HIRA complex subunits (Tubulin, loading control), and by new H3.3-SNAP labelling for H3.3. d Nascent transcript levels analysed by ethynyl-uridine incorporation (EU) as in (a). The line graphs show mean values ± s.d. from four (a) and three (d) independent experiments scoring at least 81 cells per experiment. Statistical significance is calculated by two-way (a, d) and one-way ANOVA (b) with Bonferroni posttest, and via two-sided Student’s t-test with Welch’s correction (c). ***: p < 0.001; ns: p > 0.05. Scale bars, 10 μm. Source data are provided as a Source Data file.