Figure 3. Critical roles of NF-κB activity in regulating IFNγ expression by PGE₂- and PGI₂-stimulated D1A cells.

Mouse astrocyte D1A cells were treated with PGE₂ (10 μM) in the absence or presence of the ERK1/2 inhibitor U0126 (10 μM) (A,C upper panel), KT5720 (5 μM) (C lower panel) for 24 h before extracting protein or mRNA (A,C,E,F). In select experiments, the cells were transfected with ERK1/2 or p65 siRNA before incubation with PGE₂ (10 μM) for 24 h (B,D). In separate experiments, cells were treated with PGI₂ (10 μM) for 24 h (G,H). In distinct experiments, the cells were treated with PGE₂ (10 μM) in the absence or presence of PGI₂ (10 μM) for 24 h (I–M). Total ERK1/2 (A,B), phosphorylated ERK1/2 levels (A), total NF-κB (C,D), phosphorylated NF-κB (C) and total IκB (E,G) were detected by immunoblotting using specific antibodies. Equal lane loading was demonstrated by the similar intensities of total β-actin. The nuclear and total NF-κB levels were determined by western blots (F,H,K). IFNγ protein and mRNA levels were determined by IFNγ enzyme immunoassay kits and qRT-PCR, respectively (A–D,I,J). Total amounts of protein and GAPDH served as an internal control. The luciferase activity of the IFNγ promoter was determined by dual luciferase reporter assay kits (L). The binding activity of NF-κB to the promoter of IFNγ was determined by ChIP assay (M). The data represent the means ± S.E. of atleast three independent experiments. *p < 0.05; **p < 0.01 and ***p < 0.001 with respect to the vehicle-treated or vector-transfected control. ^#p < 0.05; ^##p < 0.01 and ^###p < 0.001 compared to PGE₂-treated alone.