Effect of thiacremonone on LPS-induced NO generation, expression of iNOS and COX-2 and cell viability in RAW 264.7 cells. (a) The cells were treated with 1 μg/mL of lipopolysaccharide (LPS) only or LPS combined with different concentrations (2.5, 5, 10 μg/ml) of thiacremonone (Thia) at 37°C for 24 hours. Nitric oxide (NO) generation was determined in culture medium as described in Materials and Methods. (b) The cells were transiently transfected with an inducible nitric oxide synthetase (iNOS)-luciferase construct, and activated with LPS (1 μg/ml) alone or LPS combined with the indicated concentrations of thiacremonone for eight hours. Luciferase activity was then determined. Quantification of band intensities from three independent experimental results was determined by a densitometry, and the value under the band indicate fold difference (average) from untreated control group. (c) The cells were treated with 1 μg/mL of LPS only or LPS combined with different concentrations (2.5, 5, 10 μg/ml) of thiacremonone at 37°C for 24 hours. Equal amounts of total proteins (40 μg/lane) were subjected to 10% SDS-PAGE, and the expression of iNOS and COX-2 was detected by western blotting using specific antibodies. β-actin protein was used as an internal control. (d) RAW 264.7 cells were treated with various doses (2.5, 5, 10 μg/ml) of thiacremonone for 24 hours. Morphological changes were observed under microscope (magnification, ×200). Cell viability was determined by the CCK-8 assay described in Materials and Methods. Cells were incubated with thiacremonone in the absence or presence of LPS. Results were given in percentage related to untreated controls. All values (A, B, C and D) represent the means ± 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).