Figure 3.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) is involved in lipopolysaccharide (LPS)-induced MMP-9 expression and cell migration in RBA-1 cells. (A) Cells were pretreated with diphenyleneiodonium (DPI; 0.1, 1, and 10 μM) for 1 h and then stimulated 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 DPI (10 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 DPI (10 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 transfected with scrambled (Scrb), NOX1 or NOX2 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, NOX1, and NOX2 by Western blot, respectively. (E) Cells were pretreated with or without DPI (10 μM) for 1 h, or transfected with Scrb, NOX1 or NOX2 siRNA 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.