Figure 5.

Teuvincenone F inhibits LPS-induced proinflammatory cytokine production by suppressing NF-κB activation via decreasing ubiquitination of NEMO in THP1 cells. (A) THP1 cells were pretreated with DMSO or Teuvincenone F (25 μM) for 2 h, following stimulated with LPS (100 ng/ml) for indicated hours, immunoblot assay was used to analyze pro-IL-1β expression. (B) THP1 cells were pretreated with DMSO or Teuvincenone F (25 μM) for 2 h, following stimulated with LPS (100 ng/ml) for indicated hours and then treated with ATP (1 mM) for 45 min. ELISA was used to analyze IL-1β production. (C,D) THP1 cells were pretreated with DMSO or Teuvincenone F (25 μM) for 2 h, following stimulated with LPS (100 ng/ml) for indicated hours and ELISA was used to analyze IL-6 (C) and TNF-α (D) production. (E) THP1 cells were pretreated with DMSO or Teuvincenone F (25 μM) for 2 h, following stimulated with LPS (100 ng/ml) for indicated hours, immunoblot assay was used to analyze NF-κB and MAPKs signaling pathways. Phosphorylated p65 and total form of p65, phosphorylated JNK and total form of JNK, phosphorylated Erk and total form of Erk, phosphorylated p38 and total form of p38, came from the replicate immunoblot, phosphorylated IKKα/β and total form of IKKα/β, phosphorylated IκBα and total form of IκBα, came from the replicate immunoblot. (F) THP1 cells were pretreated with DMSO or Teuvincenone F (25 μM) for 2 h, following stimulated with LPS (100 ng/ml) for indicated hours, immunoblot assay was used to analyze NEMO. (G) THP1 cells were pretreated with DMSO or Teuvincenone F (25 μM) for 2 h, following stimulated with LPS (100 ng/ml) for indicated hours, and then subjected to immunoprecipitation with anti-NEMO antibody followed by western blot analysis with anti-Ub antibody. Similar results were obtained from three independent experiments (A,E–G). Data are shown as mean ± SD of one representative experiment in (B–D). ^*p < 0.05, ^**p < 0.01.