Figure 2

CORM-2 and CoPP/hemin-induced expression of IRG1 is dependent on HO-1 in RAW264.7 macrophages. (a) RAW264.7 cells were treated with CORM-2 (0, 5, 10 and 20 µM) or its negative control, RuCl₃ (10 and 20 µM) for 8 h and the levels of IRG1 and HO-1 mRNA were detected by using RT-PCR analysis. (b–d) Cells were pre-treated with ZnPP (10 and 20 µM) for 0.5 h and treated with CORM-2 for 8 or 16 h. The levels of IRG1 (b) and HO-1 (c) mRNA were measured at 8 h by real-time RT-PCR and (d) IRG1 protein levels were determined at 16 h by western blot analysis. (e) Cells were transfected with HO-1 siRNA or control siRNA (Con siRNA). After treatment with 20 µM CORM-2 for 16 h, cells were harvested and protein levels of HO-1 and IRG1 were performed by western blotting. (f and g) Cells were pre-treated with ZnPP (20 µM) for 0.5 h and treated with CoPP (10 µM) and hemin (10 µM) for 8 or 16 h. (f) IRG1 mRNA was measured at 8 h by real-time RT-PCR and (g) IRG1 protein levels were determined at 16 h by western blot analysis. The representative bands or blots are shown. Data represent mean±s.e.m., * P<0.05 and ** P<0.001 as compared with control and ^## P<0.001 as compared with the cells exposed to only CORM-2, hemin or CoPP.