Figure 1.

ROS generation involved in CORM-3-induced HO-1 expression. (a) RBA-1 cells were pretreated with various concentrations of NAC for 1 h and then incubated with 30 μM CORM-3 for 6 h. The levels of HO-1 and GAPDH (as an internal control) were determined by western blot. (b) The cells were pretreated with NAC (10 mM) for 1 h and then incubated with CORM-3 (30 μM) for 4 h. The levels of HO-1 and GAPDH mRNA were analyzed by real-time PCR (open bars). The cells were transiently transfected with HO-1 report gene together with a β-galactosidase plasmid, pretreated with NAC (10 mM) for 1 h, and then incubated with CORM-3 for 1 h. Promoter activity was determined in the cell lysates (solid bars). (c, d) The cells were pretreated with or without NAC (10 mM) for 1 h and then incubated with 30 μM CORM-3 for the indicated time intervals (c) or 10 min (d). ROS generation was determined by measuring fluorescence intensity of DCF-DA. (e) The cells were pretreated with NAC (10 mM) for 1 h, incubated with 30 μM CORM-3 for the indicated time intervals (CORM-3: 0, 5, 10, 15, and 30 min; NAC/CORM-3: 10 min), and then labeled with DCF-DA and DHE, respectively. The fluorescence intensity was observed under a fluorescence microscope. Data were expressed as mean ± S.E.M. of three independent experiments. Scale bar = 50 μm. ^#P < 0.05, as compared with the control or pretreatment with inhibitor indicated in the figure.