Figure 5

Effect of thiacremonone on LPS-induced NF-κB activation in RAW 264.7 and THP-1 cells. (a) RAW 264.7 cells were transfected with p-NF-κB-Luc plasmid (5× nuclear factor (NF)-κB), and then treated with lipopolysaccharide (LPS; 1 μg/ml) alone or in combination with thiacremonone (Thia; 2.5, 5, 10 μg/ml) at 37°C for six hours. Luciferase activity was then determined as described in Materials and Methods. (b) The DNA-binding activity of NF-κB was investigated using electromobility shift assay (EMSA) as described in Materials and Methods. Nuclear extracts from RAW 264.7 cells with LPS alone (1 μg/mL) or in combination with thiacremonone (2.5, 5, 10 μg/ml) were subjected to DNA-binding reactions with ³²P end-labeled oligonucleotide specific to NF-κB. The specific DNA-binding activity of NF-κB complex is indicated by an arrow. (c) For competition assays, nuclear extracts from RAW 264.7 cells treated with LPS (1 μg/ml) were incubated for one hour before EMSA with unlabeled NF-κB oligonucleotide or labeled NF-κB oligonucleotide. For supershift assays, nuclear extracts from RAW 264.7 cells treated with LPS (1 μg/ml) were incubated for one hour before EMSA with specific antibodies against the p50 and p65 NF-κB isoforms. SS indicates supershift band. (d) Cells treated with 1 μg/mL of LPS only or LPS plus different concentrations (2.5, 5, 10 μg/ml) of thiacremonone at 37°C for one hour. Equal amounts of total protein (40 μg) were subjected to 10% SDS-PAGE. Nuclear translocation of p50 and p65, and degradation of inhibitory (I) κB were detected by western blotting using specific antibodies. β-actin protein was used as an internal control. (e) Nuclear extracts from RAW 264.7 cells with another inducer alone (TNF-α (10 ng/ml), IL-1α (10 ng/ml), IFN-γ (10 ng/ml)) or in combination with thiacremonone (10 μg/ml) were subjected to DNA-binding reactions with ³²P end-labeled oligonucleotide specific to NF-κB. The specific DNA-binding of NF-κB complex is indicated by an arrow. (f) Nuclear extracts from THP-1 cells with LPS alone (1 μg/mL) or in combination with thiacremonone (2.5, 5, 10 μg/ml) were subjected to DNA-binding reactions with ³²P end-labeled oligonucleotide specific to NF-κB. The specific DNA-binding of NF-κB complex is indicated by an arrow. Values (A, B and C) are mean ± standard deviation of three independent experiments performed in triplicate. ^# indicates significantly different from control group (P < 0.05). * indicates significantly different from the LPS-treated group (P < 0.05).