ETYA-induced CCL2/MCP-1 suppression depends on the inhibition of JNK-AP1 signals. (A) Astrocytes were transiently transfected with the indicated 5'-deleted CCL2/MCP-1 promoter constructs and incubated for 48 h. After stimulation with IFN-γ in the presence of the indicated activators for 6 h, cells were harvested and luciferase activity was measured and plotted as fold-induction over untreated controls. Data are presented as means ± SEMs for three independent experiments (*p < 0.05 versus IFN-γ group). (B) Astrocytes were stimulated with IFN-γ in the presence of the indicated PPAR-α activators, and nuclear factor binding activities of AP1 were measured by EMSA using oligonucleotide probes specific for the AP1 site of the rat CCL2/MCP-1 promoter (-129 to -111). The arrows represent specific DNA-protein complexes. (C) Astrocytes were treated with ETYA or WY-14643 and prepared for ChIP assays (see Materials and Methods for details). "Input" indicates control PCR and shows the amount of CCL2/MCP-1 promoter DNA present in each sample before ChIP. (D) After stimulating astrocytes with IFN-γ for 2 h in the presence of ETYA or WY-14643, JNK activity was measured by Western blot analysis using an anti-phospho-JNK antibody. The bar graph represents the intensities of pJNK bands normalized against those of α-tubulin (bottom panel). The data are presented as means ± SDs of three independent experiments. *p < 0.05, NS; non significant.