Figure 7
Abolition of the inhibitory effect of thiacremonone by DTT and glutathione GSH, and in the cells harboring mutant p50 on NO generation and DNA binding activation of NF-κB. (a) RAW 264.7 cells grown in six-well plates were cotreated with indicated concentrations of dithiothreitol (DTT) (100 nM) or glutathione (GSH; 100 μM) with thiacremonone (Thia; 10 μg/ml) for one hour. Nuclear extracts were then prepared and examined by electromobility shift assay (EMSA) as described in Materials and Methods. (b) The cells were transiently transfected with nuclear factor (NF)-κB-luciferase construct, and were co-treated with indicated concentrations of DTT (1 to 100 nM) or GSH (1 to 100 μM) with thiacremonone (10 μg/ml) for eight hours, and then the luciferase activity was determined. (c) The cells were co-treated with indicated concentrations of DTT (1 to 100 nM) or GSH (1 to 100 μM) with 1 μg/mL of lipopolysaccharide (LPS) only or LPS plus thiacremonone (10 μg/ml) at 37°C for 24 hours. Nitric oxide (NO) generation was determined in culture medium as described in Materials and Methods. (d) The cells were transiently transfected with inducible nitric oxide synthetase (iNOS)-luciferase construct, and were co-treated with indicated concentrations of DTT (1 to 100 nM) or GHS (1 to 100 μM) with thiacremonone (10 μg/ml) for eight hours, and then the luciferase activity was determined. (e) RAW 264.7 cells were transfected with p50 mutant (C62S) plasmid at 37°C for six hours, and then NF-κB DNA-binding activity was determined after one hour of treatment with thiacremonone by electromobility shift assay (EMSA) as described in Materials and Methods. (f) NO generation was determined in culture medium as described in Materials and Methods. RAW 264.7 cells were transfected with p50 mutant (C62S) plasmid at 37°C for six hours, and then NO generation was determined after 24 hours treatment with thiacremonone as described in Materials and Methods. All values represent the means ± standard deviation of three independent experiments performed in triplicate. # indicates significantly different from control group (P < 0.05). * P < 0.05 indicate statistically significant differences from the LPS-treated group.