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. 2011 Jan 18;286(11):9856–9864. doi: 10.1074/jbc.M110.196790

FIGURE 8.

FIGURE 8.

Cellular NAD+ is required for SIRT1 promoter binding and repression of TNF-α transcription. A, cellular NAD+ is diminished by the Nampt-specific inhibitor FK866. THP-1 cells were pretreated for 24 h with 10 nm FK866. Cells were washed and stimulated for indicated times with 1 μg/ml LPS in the presence of 10 nm FK866. B, depletion of cellular NAD+ by FK866 augments LPS-induced TNF-α transcription. THP-1 cells were pretreated for overnight with 10 nm FK866 followed by stimulation for 1 h with 1 μg/ml LPS. TNF-α mRNA was quantified using real-time PCR. C, depletion of cellular NAD+ inhibits SIRT1 binding to the TNF-α promoter. THP-1 cells were treated as in A. Cell lysates were subjected to SIRT1 ChIP assay at the TNF-α proximal promoter. D, depleting NAD+ by FK866 simultaneously with inhibiting SIRT1 activity enhances accumulation of acetylated histone H4 at lysine 16. Cells were treated for 16 h with LPS in the presence or absence of 10 nm FK866 and 1 mm nicotinamide followed by ChIP analysis with H4K16Ac antibody. Med, medium; Nic, nicotinamide.