Figure 8.

PGE₂-stimulated Nurr1 protein expression (A) and mRNA expression (B); PGE₂-stimulated phosphorylation of CREB and ATF-1 (C); and PGE₂-stimulated Nurr1 responsive (NBRE) luciferase reporter gene activity (D) in SH-SY5Y neuroblastoma cells in the presence and absence of the EP₁ receptor antagonist, SC-51322 and various signalling pathway inhibitors. (A) SH-SY5Y cells were pretreated with either vehicle (control) or 1 µM SC-51322 for 30 min at 37°C and were then incubated with 1 µM PGE₂ for the indicated times. Lysates were prepared and subjected to immunoblot analysis using antibodies against Nurr1 or vinculin. (B) SH-SY5Y cells were incubated with 1 µM PGE₂ at 37°C for the indicated times and then RNA was isolated and used for quantitative real-time PCR with primers specific for either Nurr1 or GAPDH as described in the methods section. Data were analysed by the comparative ΔΔCt method, relative to the expression of GAPDH. Data are the means ± SEM (n= 4) of the pooled data from two independent experiments, each in duplicate. (C) SH-SY5Y cells were pretreated with either vehicle (control) or 1 µM SC-51322 for 30 min at 37°C and were then incubated with 1 µM PGE₂ for indicated times. Lysates were prepared and subjected to immunoblot analysis with antibodies against either phospho-CREB/ATF-1 (p-CREB/p-ATF-1) or CREB. (D) SH-SY5Y cells were transiently transfected with an NBRE luciferase reporter plasmid and ∼18 h later were pretreated with vehicle (control) or 10 µM of the NF-κB inhibitor BAY 11–7082 (BAY) or 10 µM of the PKA inhibitor H89 or 1 µM SC-51322 (SC) for 30 min at 37°C, followed by treatment with either vehicle (veh) or 1 µM PGE₂. Luciferase activity was determined the next day. Data are the means ± SEM of quadruplicate measurements from a representative experiment that was repeated three times. ***P < 0.001; *P < 0.05 compared with time 0 or with the corresponding vehicle control; one-way anova, followed by Bonferroni post-test. Immunoblots are representative from one of at least three independent experiments.