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. 2020 Aug 17;10:13907. doi: 10.1038/s41598-020-70874-6

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© The Author(s) 2020

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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The low levels of PARP-1 activity in CDA-deficient cells are rescued by the overexpression of wild-type NAMPT (a) Representative immunofluorescence deconvoluted z-projection images showing DAPI and His-tag staining in HeLa-Ctrl_(CDA) cells not transfected (NT) or transiently transfected with pPM-C-His empty vector (EV), or with a pPM-C-His construct expressing wild-type NAMPT (NAMPT WT) or mutated NAMPT (NAMPT H247A). Nuclei were visualized by DAPI staining (blue) and the His-tag was visualized with Alexa Fluor 555 (red). Scale bar: 5 µm. (b) Percentage of His-tag-positive cells among HeLa-Ctrl_(CDA) and HeLa-shCDA cells transiently transfected with EV, NAMPT WT or NAMPT H247A. The data shown are means ± SD from four independent experiments. (c) PARP-1, NAMPT, NAMPT-HIS and CDA proteins levels assessed by immunoblotting in HeLa-Ctrl_(CDA) and HeLa-shCDA cells transiently transfected with EV, NAMPT WT or NAMPT H247A. (d) Relative number of PAR foci in HeLa-Ctrl_(CDA) and HeLa-shCDA) cells transiently transfected with EV, NAMPT WT or NAMPT H247A. The data shown are means ± SD from four independent experiments (> 800 cells per condition). (e) Mean number of UFBs per anaphase cell, for HeLa-Ctrl_(CDA) and HeLa-shCDA cell lines transiently transfected with EV, NAMPT WT or NAMPT H247A. Error bars represent means ± SD from three independent experiments (> 120 anaphase cells per condition). The significance of differences was assessed in Student’s t-tests. (f) (1) CDA deficiency leads to (2) intracellular dC/dCTP accumulation that (3) decreases NAMPT activity, directly or indirectly, leading to the (4) intracellular accumulation of an as yet unidentified factor X (5) lowering basal PARP-1 activity, causing (6) excess UFB formation.