Figure 5.

SIRT1 inhibition exacerbates the inflammatory response in PBMCs. (A) Transcriptional activities of NF-κB were analyzed by luciferase reporter assay (n = 3). (B–K) PBMCs were treated with DMSO or SIRT1-inhibitor Ex527 (40 μM) for 24 h. (B) The acetylation of P65 at K310 was determined by Western blotting (n = 3). Band intensities of Ac-P65 normalized for the corresponding P65 intensity were calculated (n = 3). (C) Immunostaining showed the nuclear translocation of P65 (red) when PBMCs were treated with Ex527 (n = 3). Scale bars, 20 μm. (D–G) PBMCs were stimulated with LPS (50 ng/mL) for the last 5-h incubation. The mRNA expression levels of TNF-α (D), IL-6 (E), IL-8 (F) and MCP1 (G) were quantified by qRT-PCR (n = 4). (H–K) PBMCs treated with Ex527 were cultured with 50 ng/mL LPS for the last 5 h, and TNF-α (H), IL-6 (I), IL-8 (J) and MCP1 (K) concentrations in the culture supernatants were analyzed by ELISA (n = 3). Data represent means ± s.e.m. *P < 0.05, **P < 0.01.