Figure 6

Effect of CD73 knockout on the uptake of NAD⁺ precursors and on NAD⁺ biosynthesis. (A) Left panel: Immunoblot analysis of CD73 expression in MCF-7/Cas9 and MCF-7/CD73-KO cells; actin is shown as a loading control. Right panel: Comparative analysis of intracellular NAD⁺ levels in both cell lines exposed to different NAD⁺ precursors (100 μM) in the absence or presence of the NAMPT inhibitor, FK866 (30 nM). (B) NMR analysis to define the distribution of NAD⁺ and NAD⁺ metabolites (100 μM) in cell culture media 6 hrs after supplementation of NMN or NAD⁺ to cell-free serum-free media (SFM), cell-free media supplemented with fetal bovine serum (FBS) or when added to SFM or FBS in the presence of MCF-7/Cas9 or MCF-7/CD73-KO cells in the presence or absence of FK866 (30 nM). (C) NMR analysis to define the distribution of NAD⁺ and NAD⁺ metabolites in cell culture media 24 hrs after supplementation of NMN or NAD⁺ to MCF-7/Cas9 cells in the presence or absence of FK866. Cells were exposed to media supplemented with fetal bovine serum (FBS) or heat inactivated fetal bovine serum (FBS-HI) and compared to NMN and NAD⁺, which have never been exposed to cells.