Fig. 4

CD38 regulates extracellular but not intracellular NAD⁺ levels in RPWE1 cells. a Western blots demonstrate doxycyline (Dox) induced expression of wild-type (WT) or mutant (E226Q) CD38 in RWPE1 cells. Tubulin serves as a loading control. b Cell proliferation assay over 4 days in culture in the presence or absence of 20 ng/mL Dox. Relative cell number was assessed by measuring DNA fluorescence at 465 nm. 3–6 replicate wells per group per time point were measured. Plot shows mean ± standard error of the mean (SEM). c, d NAD⁺ and NADH levels were measured relative to total protein in each sample and presented relative to no Dox (non-induced) sample. Mean ± SEM of four replicates is shown. e NAD⁺:NADH ratio is calculated based on results shown in c and d. Mean ± SEM of four replicates is shown. f Cells were treated with Triton X-100 (TX-100) to permeabilize cells followed by NAD⁺ measurements. NAD⁺/protein is shown relative to no Dox. Mean ± SEM of four replicates is shown. g–i RWPE1 cells were treated with increasing concentrations of FK866 followed by NAD⁺ (g) and NADH (h) measurements. Mean ± SEM of four replicates is shown. Newman-Keuls Multiple Comparison Test. i Cell proliferation assay over 4 days in culture in the presence of the indicated concentrations of FK866. DNA fluorescence represents relative cell number. 3–6 replicate wells per group per time point were measured. Plot shows mean ± standard error of the mean (SEM). j Relative NAD⁺/protein levels in the media 30 min after the addition of 800 nM exogenous NAD⁺. Mean ± SEM of four replicates is shown