Effect of LicA on uPA transcriptional activity and NF-κB binding activity in SK-Hep-1 cells.
(A) SK-Hep-1 cells transfected with a luciferase reporter plasmid containing the promoter region of uPA were treated with increased concentrations of LicA for 24 h. Cell lysates were extracted from each treatment group, and the activity of uPA promoter was determined via luciferase activity. The plot demonstrates the relative activity of the uPA promoter from three independent experiments. (B) Nuclear extracts were also assessed by Western blotting with anti-ATF-2, NF-κB (p65), c-fos, and c-jun antibodies. Lamin B and α-tubulin were used as markers of nuclear and cytosolic fractions, respectively. (C) Results from the ChIP assay on the chromatin isolated from SK-Hep-1 cells. Cells were treated with different concentration of LicA and immunoprecipitated with anti-NF-κB (p65) or control IgG. The isolates were assessed with PCR using primers targeting the sequence on the uPA promoter. The bottom plot shows the relative quantitative results compared to that of the input. (D) The nuclear localization of NF-κB after treatment with or without LicA and/or SP600125, respectively. (E) The effects of si-MKK4 and LicA on the nuclear translocation of NF-κB, cells were pretreated with si-MKK4 for 6 h and then incubated in the presence or absence of LicA (10 µM) for 18 h. Afterward, the nuclear extracts were also assessed by Western blotting to analyze the nuclear translocation of NF-κB. Bars represent the mean±S.E. from three independent experiments. *p<0.01, compared with the Input.