Pharmacological inhibition of PNP blocks NR conversion to Nam in various types of mammalian cells. A–F, HEK293, HeLa, THP1, A549 cells, or HDF (human dermal fibroblasts) were treated with 150 μM NR and Immucillin H (ImmH). mESC E14 cells were treated with 100 μM NR and ImmH. A, 24 h before the treatment, HEK293 cells were transiently transfected with a vector encoding FLAG-tagged ENT1. Left panel, schematic representation of the experimental approach. Twenty-four hours after the treatment, culture media were analyzed by NMR spectroscopy. Levels of NR and Nam in culture medium are presented. G, 2 × 109 of isolated human red blood cells (RBCs) in 1 ml Hepes buffer were incubated with NR (300 μM) and ImmH for 2 h. The supernatant was then collected and analyzed by NMR as above. Relative levels of extracellular NR and Nam are presented. The concentration of NR in control buffer incubated without cells was taken as 100%. Data are presented as mean ± S.D. (n = 3). nd, not detected. Statistical analysis of differences between the groups was carried out by one-way ANOVA with post hoc comparisons using the Tukey test. ∗ indicates statistical significance at p < 0.05, ∗∗ indicates statistical significance at p < 0.01, ∗∗∗ indicates statistical significance at p < 0.001. ENT, equilibrative nucleoside transporter; Nam, nicotinamide; NBTI, S-(4-nitrobenzyl)-6-thioinosine; NMN, Nam mononucleotide; NR, nicotinamide riboside; PNP, purine nucleoside phosphorylase.