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. 2019 Dec 16;2019:6879346. doi: 10.1155/2019/6879346

Figure 2.

Figure 2

Anti-inflammatory effects of Tt-ME on gene expression of cytokines and nuclear translocation of NF-κB, AP-1, and STAT3 transcription factors. (a) RAW264.7 cells were treated with increasing concentration of Tt-ME for 30 min and then treated with LPS (1 μg/ml) for 6 h followed by isolation of RNA and generation of cDNA. Quantitative PCR levels of iNOS, TNF-α, and IL-6 gene expression were determined with GAPDH as a housekeeping gene. (b) Luciferase reporter assay. HEK293T cells were transfected over night with NF-κB, AP-1 luciferase vector, and β-gal expression vector constructs and then treated with the indicated dose of Tt-ME overnight. The levels of NF-κB and AP-1-mediated luciferase were determined by using a luminometer. (c) RAW264.7 cells were pretreated with Tt-ME (200 μg/ml) for 30 minutes and then treated with LPS (1 μg/ml) for the indicated period of time. Representative western blot analyses of NF-κB subunits (p50 and p65), AP-1 subunits (c-Fos and c-Jun), and STAT3 were performed, and the nuclear translocation levels were calculated by densitometric analysis of band intensities. Data represent mean ± SD, replicates of three (∗p < 0.05; ∗∗p < 0.01 students t-test). The experiment was repeated, and similar results were observed.