Figure 1

Pristimerin abrogated TNF-induced NF-κB activation at TAK1 and IKK steps. (A) Structure of pristimerin. (B and C) Pristimerin inhibited TNFα-induced NF-κB-dependent reporter gene expression. U2OS cells were cotransfected with 60 ng NF-κB-TATA-Luc reporter plasmid and 10 ng Renilla luciferase reporter plasmid. 24 hours later, cells were treated with different concentrations of pristimerin (B) or a fixed concentration (200 nM) for various durations (C), then TNFα (0.1 nM) for 10 minutes; cell supernatants were prepared, and luciferase intensity was measured. The levels of firefly luciferase activity were normalized to Renilla luciferase activity. Results were expressed as fold change ± SE of at least 3 independent experiments. ***, P < 0.0001, one-way ANOVA, post hoc comparisons, Tukey's test. Columns, mean; error bars, SE. (D) Pristimerin abrogated IKK-induced NF-κB activation. U2OS cells were cotransfected with the indicated plasmids (p65, IKKα, IKKβ, IKKγ) along with NF-κB-TATA-Luc reporter plasmid. 24 hours later, cells were treated with 200 nM pristimerin for 6 hours. The expression of Renilla luciferase activity normalized transfection efficiency. *, P < 0.05; **, P < 0.01; ***, P < 0.0001, one-way ANOVA, post hoc comparisons, Tukey's test. Columns, mean; error bars, SE. (E) Pristimerin diminished TAK1-induced NF-κB activation. U2OS cells were cotransfected with the TAK1-expressing plasmid and NF-κB-TATA-Luc reporter plasmid. 24 hours later, cells were treated with increasing concentrations of pristimerin for 6 hours. Luciferase activity was assayed as described above. **, P < 0.01; ***, P < 0.0001, one-way ANOVA, post hoc comparisons, Tukey's test. Columns, mean; error bars, SE.