2.2 SIRT-1 pathway

We analyzed the effect of a SIRT-1 activator SRT1720 [32]. Regarding dose-response experiments [33], at first, we evaluated concentrations between 0 and 10 μM for 48h. However, since the higher concentrations (2.5–10 μM) induced cell toxicity, we further analyzed the effect of SRT1720 at concentrations 0.5, 1 and 2 μM. The absence of toxicity was evaluated by visual inspection and by the verification that the total protein amount in each dish was equivalent to that of the control at the end of the experiment. The efficacy of SRT1720 was evaluated by its ability to inhibit NFκB. We determined that the concentration of 2 μM was optimal to induce SIRT-1 and inhibit NFκB. We also evaluated the effect of a SRTI-1 inhibitor, splitomicin [34]. We tested splitomicin at concentrations ranging from 0 to 500 μM according to the literature[35]. We discarded the 500 μM concentration which was toxic and evaluated the ability of splitomicin to induce NFκB at concentrations of 50, 100, 200 μM. The dose-response evaluation allowed to determine that the concentration of 100 μM was optimal to decrease SIRT-1 and to activate NFκB. Thereafter, these concentrations of SRT-1720 and splitomicin were used in all the experiments performed. The cells were cultured during 15 days with/without the antiretroviral molecules, SRT1720 or splitomycin being added for the last 3 days.