Figure 6

NF-κB is involved in TGF-β1-induced MMP-9 expression and cell migration in RBA-1 cells. (A) Cells were treated with TGF-β1 (15 ng/ml) for 16 h in the absence or presence of helenalin or Bay11-7082. (B) Cells were pretreated with helenalin (HLN, 1 μM) or Bay11-7082 (Bay, 1 μM) before exposure to TGF-β1 for 6 h. The conditioned media and total RNA were collected and analyzed by gelatin zymography (A) and RT-PCR (B). (C) Time dependence of TGF-β1-stimulated NF-κB p65 phosphorylation, cells were incubated with TGF-β1 (15 ng/ml) for the indicated time intervals. (D) Cells were pretreated with U0126 (U0, 10 μM), SP600125 (SP, 10 μM), NAC (100 μM), or Bay11-7082 (Bay, 1 μM) for 1 h before exposure to TGF-β1 for 1 h. Whole cell lysates were analyzed by western blotting using an anti-phospho-NF-κB-p65 antibody. (E) For cell migration, cells were pretreated with Bay11-7082 (1 μM) for 1 h and then incubated with TGF-β1 (15 ng/ml) for 48 h. Representative phase contrast images are shown for 48 h (n = 3). Data are expressed as mean ± SEM (C) or mean (A, B, D) of three independent experiments (n = 3). *P < 0.05; ^#P < 0.01, as compared with the cells exposed to vehicle (C) or TGF-β1 (A, B, D) alone. The figure represents one of three similar experiments.