Figure 5

ROS-dependent MAPK signaling is essential for 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 N-acetyl cysteine (NAC). (B) Cells were pretreated with NAC (100 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 intracellular ROS generation, RBA-1 cells were incubated with the peroxide-sensitive fluorescent probe DCF-DA (5 μM) for 45 min, followed by stimulation with TGF-β1 (15 ng/ml) for the indicated time intervals. Moreover, cells were treated with TGF-β1 for 10 min in the presence of NAC (NAC/10, 100 μM). The DCF fluorescence intensity of cells was determined. (D) Cells were pretreated with or without NAC (100 μM) for 1 h before exposure to TGF-β1 for 10 min (for ERK1/2) or 4 h (for JNK1/2). Whole cell lysates were analyzed using an anti-phospho-ERK1/2, anti-phospho-JNK1/2, or anti-GAPDH (as an internal control) antibody. (E) For cell migration, cells were pretreated with NAC (100 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). (F) Cells were pretreated with or without NAC (100 μM) for 1 h before exposure to H₂O₂ (0.1 or 1 μM) for 16 h or H₂O₂ (100 μM) and TGF-β1 for 16 h. Data are expressed as mean ± SEM (C) or mean (A, B, D, F) 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, F) alone. The figure represents one of three similar experiments.