Figure 5.

Pharmacological or genetic suppression of PNP potentiates the NAD synthesis from NR in human cells. A–D, HEK293, HeLa or THP1 cells were treated with NR and the inhibitor of PNP, Immucillin H (ImmH) as indicated. To inhibit NAD synthesis from Nam, cells were treated with FK866. E and F, wildtype (wt) or PNP knockout (ko) HEK293 cells were treated with NR and FK866, as indicated. Twenty-four hours after the treatment, intracellular NAD levels were measured by NMR spectroscopy (A and E), and relative metabolic activity was assessed using the MTT-assay (B and F). Forty-eight hours after the treatment the fraction of dead cells was determined by flow cytometry (C and G). Twenty-four hours after the treatment, the extent of α-tubulin (K40) acetylation was estimated by immunoblotting (D); right panel, densitometric quantification of immunoblots as in left panel. Data are presented as mean ± S.D. (n = 3). nd, not detected. ns, not significant. Statistical analysis of differences between the groups was carried out by one-way ANOVA with post hoc comparisons using the Tukey test. Panels A, and C–G, ∗ indicates statistical significance at p < 0.05, ∗∗ indicates statistical significance at p < 0.01, ∗∗∗ indicates statistical significance at p < 0.001. Panel B, ∗ indicates statistical difference at p < 0.001 versus the FK866-treated cells, # indicates statistical difference at p < 0.001 and ## - statistical difference at p < 0.05 versus the treatment with the corresponding concentration of NR, but without Immucillin H, + indicates statistical difference at p < 0.001 versus the treatment with 10 μM NR, but without Immucillin H. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NR, nicotinamide riboside; PNP, purine nucleoside phosphorylase.