Figure 4.

NADPH oxidase activation and ROS generation by KPR regulate HO-1 expression. (A) HPAEpiCs were pretreated with various concentrations of APO or NAC for 1 h and then incubated with vehicle or KPR (10 μM) for 16 h. The protein expression of HO-1 was determined by Western blot using GAPDH as a loading control. (B) Cells were pretreated with or without APO (100 μΜ) or NAC (1 mM) for 1 h, and then incubated with DMSO or KPR (10 μM) for 6 h or 8 h. The HO-1 mRNA expression and promoter activity were analyzed by real-time PCR (6 h) and promoter activity assay (8 h), respectively. (C,D) The chemiluminescence was measured for NADPH oxidase activation and ROS accumulation. Cells were treated with KPR (10 μM) at the indicated time intervals. (D) Cells were pretreated with APO (100 μM) or NAC (1 mM) for 1 h and then incubated with KPR (10 μM) for the indicated time intervals (30 min for NADPH oxidase activation; 2 h for ROS). (E) Cells were transfected with p47 or NOX2 siRNA, and then incubated with KPR (10 μM) for 16 h. The protein levels of HO-1, p47, NOX2, and GAPDH were determined by Western blot. (F) Cells were pretreated with p47 siRNA, and then incubated with KPR (10 μM) for the indicated time intervals. The levels of phospho-p47 and total p47 were determined by Western blot. Data are expressed as mean ± SEM (n = 5), analyzed with one way ANOVA and Dunnett’s post hoc test. # p < 0.01, as compared with the cells exposed to vehicle alone; or significantly different as indicated.