T. gondii-induced MIF expression is mediated through the activation of JNK and p38 MAPK pathway, followed by the AP-1 transcriptional activation. (A) BMDMs were infected with T. gondii RH strain (moi = 1) for the indicated time periods. Cells were harvested and subjected to western blotting analysis for phosphorylated ERK, p38, or JNK. β-tubulin served as a loading control. (B) BMDMs were pretreated with PD98059 (5, 10, 20 μM), SP600125 (5, 20, 30 μM) or SB203580 (1, 5, 10 μM) for 45 min, followed by infection with T. gondii RH strain (moi = 1) for 18 h. Immunoblot (top) or qPCR (bottom) analysis was performed to determine protein and mRNA expression MIF, respectively. (C) Raw 264.7 cells were transfected with plasmids carrying AP-1 luciferase reporter constructs before T. gondii RH strain (moi = 1, top; indicated moi, bottom) for various time periods (top) or 18 h. Luciferase assays were performed based on normalization to the β-galactosidase activity. (D) Effects on AP-1 transcriptional activity in the presence or absence of general antioxidant (NAC; 1, 2, or 5 mM), Nox inhibitor (DPI; 1, 5, or 10 μM) or superoxide scavenger (Tiron; 5, 10, or 20 mM). The experimental conditions were as outlined in Fig. 2C. Data are representative of three independent experiments and are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student’s t-test.