Figure 4.

Nicotinamide phosphoribosyltransferase (NAMPT) reduces the acetylation of H3K9 in a toll-like receptor 4 (TLR4)–dependent manner. A, Human lung endothelial cells (ECs) were pretreated with vehicle (Veh) or CLI-095 (5 μM) for 1 hour and stimulated with recombinant human NAMPT (rhNAMPT; 1 μg/mL) for 4 hours. The cells were lysed, and Western blot was performed with antibodies specific for acetyl H3K9. Total H3K9 was used as loading control. Results demonstrate the extracellular NAMPT reduced the H3K9 acetylation, which is reversed by TLR4 inhibitor. B, Densitometric summary of the attenuation of rhNAMPT-induced H3K9 acetylation by TLR4 inhibitors. rhNAMPT-induced EC demonstrated reduced H3K9 acetylation (∼65% reduction). Pretreatment with TLR4 inhibitor alone did not significantly alter H3K9 acetylation levels but prevented the rhNAMPT-induced reduction of H3K9 acetylation. Bar graphs represent data as fold-change of integrated density of ac-H3K9 normalized to pan-H3K9 for each sample. Two independent experiments were performed per condition; one asterisk indicates P  < 0.05 comparing the rhNAMPT-stimulated cells versus the TLR4 inhibitor–pretreated and rhNAMPT-stimulated cells.