Figure 4.

Inhibitory effects of deoxyelephantopin (DET) on tumour necrosis factor α (TNF-α)-induced activation of matrix metalloproteinase (MMP)-9, MMP-2 and nuclear factor-kappa B (NF-κB), and p65 translocation in TS/A cells. TS/A cells were treated with DET, then TNF-α for 24 h. In (A1) and (A2), MMP-9 and MMP-2 enzyme activity was analysed by gelatin zymography and protein expression by immunoblotting. Data are presented as mean ± SEM of three independent experiments. Different letters between treatments within the same assay indicate significant differences (one-way anova). (B) Immunofluorescent cell staining and microscopy. TS/A cells were treated with vehicle or DET for 1 h, then stimulated with TNF-α (1 ng·mL⁻¹) for 30 min. Cells were stained with 4,6-diamidino-2-phenylindole (blue), FITC-labelled anti-α-tubulin antibody (green) and rabbit anti-p65 antibody (red). (C) Electrophoretic mobility shift assay (EMSA) analysis. Nuclear extracts (8 µg) from the TS/A cells with or without stimulation with TNF-α were treated with DET for 30 min at 37°C in vitro or the cells with the same treatments in (B) in vivo were subjected to EMSA with a biotin-labelled DNA probe containing the NF-κB binding site. Arrow, NF-κB-bound DNA complex. Data are representative of three independent experiments.