Figure 7.

The overexpression of SIRT1 promotes activation of mTOR signaling and decreased autophagy in BMDMs. (A) Western blot analysis shows earlier and increased phosphorylation of S6 in SIRT^oe BMDMs after LPS. (B) Enzyme-linked immunosorbent assay of BMDM supernatants that were pretreated with 50 nM of rapamycin 1 hour before LPS treatment for 24 hours showed stronger decrease in IL-1β secretion in SIRT^oe BMDMs compared with WT cells (expressed in % of reduction). (C) Western blot analysis showing increased phosphorylation of ULK in serine 757 and increased accumulation of p62 in SIRT^oe BMDMs in response to LPS compared with WT cells. To evaluate autophagy, lysosomal proteolysis was inhibited by pretreating cells with 20 mM NH₄Cl and 100 μM leupeptin at 2 hours before LPS treatment. In these conditions, (D) Western blot analysis shows increased accumulation of lipidated LC3II in LPS/WT BMDMs compared with LPS/SIRT^oe cells that shows lower LC3II but higher LC3I expression. (E) Attenuated autophagy in LPS/ SIRT^oe BMDMs was confirmed by ICC using anti-LC3 antibody (green). (F) Further quantification shows decreased presence of fluorescent-labeled LC3 compared with the WT mice, which show increased LC3 staining 2 and 3 hours after LPS. Experiments were done twice in triplicate. Values are mean ± SEM. ∗P < .05, ∗∗P < .01 (WT vs SIRT^oe). Representative microscopical images are shown at ×20 magnification.