Figure 2

NADPH oxidase-dependent ROS generation is involved in BK-induced MMP-9 expression in RBA-1 cells. (A) Cells were pretreated without or with DPI (1 μM) for 1 h before exposure to 10 nM BK for the indicated time intervals. The conditioned media were collected for zymographic analysis of MMP-9 expression. (B) Cells were pretreated with or without DPI (1 μM) for 1 h before exposure to 10 nM BK for 16 h. The total RNA was collected and analyzed by RT-PCR. (C) Cells were pretreated without or with DPI (1 μM) for 1 h and then incubated with 10 nM BK for the indicated time intervals or 5 min. The Nox activity was analyzed. (D) The total RNA was collected and analyzed Nox isotypes expression by RT-PCR. (E) Cells were transfected with scramble (scra) or Nox2 siRNA for 24 h, followed by incubation with 10 nM BK for 24 h. The conditioned media and cell lysates were collected for zymography of MMP-9 or Western blotting to determine the levels of Nox2 and GAPDH (as an internal control). Data are expressed as the mean ± SEM (n = 3). ^*P < 0.05; ^#P < 0.01, as compared with the respective values of cells stimulated with vehicle (C) and BK (A-C, E) only. The figure represents one of three similar experiments.