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. 2020 Jan 20;10:651. doi: 10.1038/s41598-020-57506-9

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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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Assessment of the time dependent changes of extracellular NAD⁺ and NAD⁺ metabolite composition and intracellular NAD⁺ levels following metabolite supplementation and/or NAMPT inhibition. (A) NMR analysis of the composition of NAD⁺/NAD⁺-metabolites in MCF-7 cell supernatants from cells exposed to NAD⁺ for 24 hrs. (B) Measurements of intracellular NAD⁺ content in MCF-7 cells exposed to NAD supplements (NMN and NR) and/or the NAMPT inhibitor, FK866, at different time points after supplement addition. Statistical analysis was performed using GraphPad Prism 7 - one-way ANOVA followed by post-hoc test with Tukey’s correction was used. (ns = not significant, ***p = 0.0004 or ****p < 0.0001) (C) Immunoblot analysis to evaluate changes in CD73 expression impacted by the treatment of MCF-7 cells with FK866 (30 nM), NMN (100 μM) or NAD⁺ (100 μM) for times ranging from 0.5 to 24 hrs, as compared to the untreated control, as indicated.