FIGURE 3.

SIRT1 represses TLR4-induced TNF-α transcription in normal and endotoxin-tolerant cells. A, effect of SIRT1-specific inhibitor Ex527 on TNF-α transcription. Normal THP-1 cells were pretreated with 1 μm Ex527 for 1 h followed by stimulation with 1 μg/ml LPS. TNF-α mRNA was quantified using real-time PCR. B, SIRT1 knockdown augments LPS-induced TNF-α transcription. Normal THP-1 cells were transfected with SIRT1-specific siRNA for 24 h as described under “Experimental Procedures.” Cells were then stimulated for 1 h with LPS. The insert shows Western blot analysis of SIRT1 protein levels. C, SIRT1 activator resveratrol inhibits LPS-induced TNF-α transcription. Normal THP-1 cells were pretreated for 1 h with 250 μm resveratrol followed by stimulation for 1 h with 1 μg/ml LPS. D, degradation of LPS-induced TNF-α mRNA. THP-1 cells were stimulated with 1 μg/ml LPS for 1 h in the presence or absence of Ex527 to induce maximal TNF-α mRNA. Cellular gene transcription was then inhibited by incubation of cells with 5 μg/ml of actinomycin D for indicated times. TNF-α mRNA levels are quantified using real-time PCR analysis and are presented as percentage of the maximum TNF-α mRNA. One of two similar experiment results is shown. SIRT1 inhibitor Ex527 (E) and SIRT1 knockdown (F) partially restore TNF-α transcription of tolerant THP-1 cells in response to second LPS stimulation. LPS-tolerant cells were treated as in A (Ex527) or as in B (SIRT1 knockdown) before LPS stimulation for 1 h. Data in A, B, C, E, and F are presented as percentage of control TNF-α mRNA and shown as mean ± S.E. Ctrl, control; KD, knockdown; Ex, Ex527; Res, resveratrol; ETOH, ethyl alcohol.