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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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Comparative analysis of CD73 expression in human cancer cell lines and its effect on NAD⁺ biosynthesis. (A) Measurement of mRNA expression for the CD73/NT5E gene in cancer cell lines, as determined by qRT-PCR analysis, normalized to the expression of human β-actin mRNA via the ∆∆CT method. (B) Immunoblot analysis of the expression of CD73 in nine breast cancer cell lines, including the MCF-7 and MDA-MB-231 cells used herein (top panel) and two glioma cell lines, including the LN428 and T98G cells used herein (bottom panel). PCNA was used as a loading control for the top panel and actin was used as a loading control for the bottom panel. See Supplement Fig. S2F for the expression of NRK1 in the breast cancer cell lines analyzed from the same cell lysates. (C) Total intracellular NAD levels (NAD⁺ and NADH) in each of the four cancer cell lines cultured in the presence of NAD⁺, NMN or NR (100 μM) for 24 hrs. Statistical analysis was performed using GraphPad Prism 7 and two-way ANOVA followed by post-hoc test with Tukey’s correction (ns = not significant, *p = 0.0419, **p = 0.0032).