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. 2018 Jan 17;9:250. doi: 10.1038/s41467-017-02293-7

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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. 1 — NUDT5 is a key regulator of ADP-ribose metabolism. a Hydrolysis of potential oxidized nucleotides and nucleotide-sugar substrates by MTH1 (blue) and NUDT5 (red), as measured by the enzyme-coupled malachite green assay (MG assay), at pH 7.5. A representative experiment (of n = 2) with mean ± SD and individual values of quadruplicate replicates is shown. b Representative HPLC traces of NUDT5-mediated ADPR (red) and 8-oxo-dGDP (blue) hydrolysis to AMP or 8-oxo-dGMP, respectively (n = 2). c Top: Representative, pseudo-colored immunofluorescence stainings for γH2A.X, chromatin-bound RPA, and 53BP1 in U-2 OS cells following treatment with negative control siRNA (siNeg) or NUDT5 siRNA (siNUDT5 #1, siNUDT5 #7) for 72 h. Scale bar = 20 µm. Bottom: Quantification of γH2A.X, chromatin-bound RPA, and 53BP1 staining intensity in cells representing three independent experiments (cells scored: γH2A.X, n = 871; RPA, n = 463; 53BP1, n = 981). Lines represent median fluorescence (and interquartile range for 53BP1); a.u. arbitrary units. NS not significant, ***p < 0.001, ****p < 0.0001; Kruskal-Wallis test). d Top: Representative, pseudo-colored images of treated U-2 OS cells following the modified alkaline comet assay and confirmation of NUDT5 knockdown with a cropped western blot. Scale bar = 50 µm. Bottom: Quantification of the tail moment (relative to the DMSO+siNeg, buffer-treated control) from two independent experiments performed in duplicate (minimum of 100 cells per slide per condition). Buffer-treated samples are shown in blue, OGG1-treated samples in red. Lines represent median tail moments. ****p < 0.0001 by Kruskal–Wallis test comparing OGG1-treated samples. e A graphical procedure depicting the analysis of ADPR hydrolysis in cell lysates by HPLC. f ADP-ribose hydrolysis in U-2 OS cells alone (black, solid and dotted), following depletion with two different NUDT5 siRNAs (blue, solid and dotted) or siRNA-treated cells complemented with purified NUDT5 protein (red, solid and dotted), as quantified by HPLC. A representative chromatogram is shown (of n = 2 independent experiments) and NUDT5 knockdown confirmation with cropped western blot