Figure 2

HTS of the NIH Clinical Collection module in N2a-E4 cells and effects of A03, fluoxetine, and memantine. (a) SirT1 levels in N2a cells stably transfected with ApoE4 (N2a-E4) are approximately half those in N2a cells stably transfected with ApoE3 (N2a-E3). (AU = Arbitrary Units; n = 3 wells per condition; p = 0.0001; statistical analysis performed using an unpaired two-tailed t-test). (b) As part of optimization and refinement of our HTS, we used N2a-E4 cells to screen the NIH Clinical Collection module comprising ~720 compounds and including multiple representations of A03 (32 per plate; −3 plates total). The mean value of SirT1 levels after A03 treatment is represented by the horizontal line, and mean value of SirT1 levels after DMSO control treatment is within the bracketed dotted lines. A03, but not SSRI fluoxetine or NMDA receptor antagonist memantine (each n = 1), increased SirT1 in N2a-E4 cells over control levels; however, because fluoxetine and memantine were only represented once in HTS, a secondary assay was performed to confirm these findings. (c) Protein concentrations in lysates used for assay from N2a cells treated with DMSO vehicle or A03 at 5 or 50 μM were very similar. (d) SirT1 (normalized to protein) showed a dose-response increase with A03 treatment (p = 0.0443 and <0.0001 for 5 and 50 μM, respectively). Statistical analysis was performed using one-way ANOVA and Tukey’s multiple comparison test; for the ANOVA summary F = 58.47 and p = < 0.0001. (e) SirT2 (normalized to protein) was not significantly altered by A03; the ANOVA summary was 0.8345 and p = 0.4651 (For protein, SirT1 and SirT2, n = 12, 3, and 3 for DMSO, 5 and 50 μM A03, respectively). Results graphed as mean ± SEM.