ROS are essential for LTA-induced MMP-9 expression in RBA-1 cells. a Cells were pretreated with or without N-acetylcysteine (NAC, 10 mM) for 1 h before exposure to 50 μg/ml LTA for the indicated time intervals. The conditioned media were analyzed by gelatin zymography for MMP-9 expression and activity. b Cells were treated with or without NAC (10 mM) for 1 h before exposure to 50 μg/ml LTA for 16 h. The total RNA was analyzed by RT-PCR (upper panel) and real-time RT-PCR (lower panel). c Cells were incubated with the peroxide-sensitive fluorescent probe DCF-DA (5 μM) for 45 min, followed by stimulation with 50 μg/ml LTA for the indicated time intervals or for 15 min in presence or absence of 10 mM NAC. The fluorescence intensity of cells was determined. d Rat primary astrocytes were isolated and cultured, and pretreated with or without NAC (1 or 10 mM) before exposure to LTA for 16 h. The conditioned media were analyzed by gelatin zymography (upper panel). For cell migration, cells were plated on coverslips and grown to confluence, then the coverslips were transferred to a new 10-cm dish containing serum-free medium for 24 h. Cells were pretreated with MMP2/9 inhibitor (2/9i) or NAC for 1 h and then incubated with LTA (50 μg/ml) for 48 h. Phase contrast images of cells were taken. The number of LTA-induced cell migrations was counted (lower panel). Data are expressed as the mean ± SEM of three independent experiments. *
P < 0.05; #
P < 0.01, as compared with the respective values of cells stimulated with vehicle (c) or LTA alone (c, d). The figure represents one of three similar experiments.