Figure 2.

P47^phox is involved in lipopolysaccharide (LPS)-induced MMP-9 expression and cell migration in RBA-1 cells. (A) Cells were pretreated with apocynin (APO; 1, 10, and 30 μM) for 1 h and then incubated with LPS (2 μg/mL) for 24 h. The levels of MMP-9 were examined by gelatin zymography. The GAPDH level of cell lysates was assayed by Western blot. (B) Cells were pretreated with APO (30 μM) for 1 h and then incubated with LPS (2 μg/mL) of 4 h for mRNA expression or 6 h for promoter activity. The mRNA expression and promoter activity of MMP-9 were determined by real-time PCR and promoter assay, respectively. (C) Cells were pretreated with or without APO (30 µM) for 1 h and then incubated with LPS (2 μg/mL) for 10 min. The fluorescence intensity of DCFH-DA or DHE staining was detected by a fluorescence microscope. The figure represents one of three individual experiments. Scale bar = 50 µm. (D) Cells were individually transfected with scrambled (Scrb) or p47^phox siRNA and then incubated with LPS (2 μg/mL) for 24 h. The medium and cell lysates were collected to determine the levels of MMP-9 by gelatin zymography and the levels of GAPDH and p47^phox by Western blot, respectively. (E) Cells were pretreated with or without APO (30 μM) for 1 h (left panel), and transfected with Scrb or p47^phox siRNA (right panel) and then incubated with LPS (2 μg/mL) for 48 h. The number of cell migration was determined (magnification = 40×). Data are expressed as mean ± SEM of three independent experiments. # p < 0.01 as compared with the cells exposed to vehicle or LPS, as indicated.