Figure 3

TNF-α induced NF-κB activation and 3-MA attenuated the activation of NF-κB. Hep3B and SMMC-7721 cells were cultured under serum starvation condition for 6 h, and 3-MA was added to the cell culture at the same time. Then cells were treated with or without TNF-α (10 ng/ml) for 24 h. (A and B) Total cellular protein(50 μg) was separated by SDS-PAGE and immunoblotted for IκBα, P65 and GAPDH. (C and D): Hep3B and SMMC-7721 cells were stimulated with TNF-α in the presence of 3-MA for the indicated times (hours). Western boltting was performed with anti-IκBα. (E and F) Cells were transduced using an NF-κB luciferase construct, then the cells were treated according to the previously described steps. At the end of the various treatment, firefly and renilla luciferase activities were assessed using a dual luciferase reporter gene assay kit. (G) NF-κB nuclear translocation was assessed by immunofluorescence staining for NF-κB p65 (green), cell nuclei were detected by DAPI (× 400). The percentage of the p65 nuclei-positively stained cells to the total cells was quantified. Data are presented as the mean ± SEM from three independent experiments. **P < 0.01; Student’s t-test.