FIGURE 1.

Extracellular micromolar NAD⁺ improves cell viability and increases intracellular NAD⁺ content in FK866-treated cells. A, A549, PC3, and U87 cell viability was evaluated by a sulforhodamine B colorimetric assay (SBC) after a 72-h incubation of cells in complete medium in the presence (or not) of 30 nm FK866, with the addition (twice a day) of 0, 0.15, 0.30, 0.6, 1.2, 2.5, 5, 10, or 20 μm NAD⁺. Results are expressed as a percentage of cell growth relative to untreated cells. In the absence of NAD⁺, FK866 treatment resulted in 8.6 ± 0.4, 11.9 ± 0.4, and 11.4 ± 1.4% cell viability in PC3, A549, and U87 cells, respectively. EC₅₀ values for the NAD⁺-rescuing activity were calculated from the non-linear regression curves with GraphPad Prism version 5. R² values were 0.981 for PC3, 0.993 for A549, and 0.966 for U87 cells. B, A549, PC3, and U87 cells were treated for 72 h with or without 30 nm FK866 in the presence or absence of 10 μm NAD⁺ (added twice a day). Cells were harvested and lysed in 0.6 m PCA, and NAD⁺ content was measured in neutralized extracts. NAD⁺ values, normalized to protein content, are expressed as relative to untreated, control cells. Data are expressed as mean ± S.D. (error bars) (n = 3). The intracellular NAD⁺ content was 6.2 ± 1.1, 7.1 ± 0.9, and 11.6 ± 1.9 nmol/mg protein in A549, PC3, and U87 cells, respectively. *, p < 0.05; #, p < 0.01; §, p < 0.001 compared with the corresponding FK866-treated cells. C, A549, PC3, and U87 cell viability was evaluated by SBC after a 72-h incubation of the cells in complete medium without (control) or with 30 nm FK866 or with FK866 supplemented with either 1 mm octanol or 10 μm NAD⁺ (added twice a day). Results are expressed as a percentage of cell growth relative to untreated cells. Data are expressed as mean ± S.D. (n = 3). No statistical difference was observed between cells treated with FK866 and NAD⁺ in the presence or absence of octanol. D, A549, PC3, and U87 cell viability was evaluated by SBC after a 72-h incubation of the cells in complete medium with or without 30 nm FK866, 10 μm Gap26, or a scrambled peptide (scr) and 10 μm NAD⁺ (added twice a day). Results are expressed as a percentage of cell growth relative to untreated cells. Data are expressed as mean ± S.D. (n = 3). No statistical difference was observed between cells treated with FK866 and NAD⁺ in the presence or absence of scrambled peptide or Gap26.