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. 2019 May 13;10:2135. doi: 10.1038/s41467-019-10153-9

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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. 6 — DilncRNA degradation is necessary for RPA recruitment. a The EXOSC10 catalytically inactive mutant EXOSC10^dea or wild-type EXOSC10 were overexpressed in HeLa cells and γH2AX immunofluorescence was performed at 0, 1, 6, and 24 h after exposing the cells to γ-radiation (5 Gy). The graph shows the percentage of cells with γH2AX foci (n = 150 cells analysed at each time point, from three different experiments). b HeLa cells were transfected with either siCtrl or siEXOSC10. A second transfection was carried out 24 h later with plasmids for expression of EXOSC10^wt or EXOSC10^dea, as indicated. The cells were micro-irradiated with a 365 nm UV laser 24 h after the second transfection and the recruitment of RPA to the laser stripes was analysed as in Fig. 2b (n > 20 cells for each condition, from three independent experiments). c HeLa cells were transfected with either siCtrl or siEXOSC10. A second transfection was carried out 24 h later with a plasmid for expression of RNase H1 and the recruitment of RPA to laser stripes was analysed (n = 50 cells analysed in each condition, from three independent experiments). d HeLa cells transfected with either siCtrl or siEXOSC10 were micro-irradiated with a 365 nm UV laser, permeabilized with CSKT and treated with or without RNAse A for 30 min at 37 °C. The graph shows the percentage of cells with γH2AX stripes that were co-stained with RPA (n > 80 cells analysed in each condition, from three independent experiments). e, f SMART was done in U2OS cells transfected with either siCtrl or siEXOSC10. The cells were treated with 10 µM Triptolide for 30 min (e) or 8 µM ActD for 1 h (f) before exposure to γ-radiation (10 Gy). The plot shows the measurements of fibre length from one representative experiment (n = 200 analysed fibres). The averages of the median values from three independent experiments are shown in Supplementary Fig. 9. In all cases, the error bars represent s.e.m. and p-values were calculated with Mann–Whitney’s test. Source data for Fig. 6a–f are provided as a Source Data file