Fig. 6
Upregulated Nur77 is involved in 6-MP-mediated inhibitory effect on TNF-α production. BV-2 cells (a, b) and primary microglia (c, d) were treated with vehicle or various concentrations of 6-MP for 1 h (a, c) or 16 h (b, d). The expression of Nur77 mRNA (a, c) was quantified by real-time RT-PCR. Nuclear extracts were prepared and subjected to western blotting (b, d) using antibody specific for Nur77. HDAC1 immunoblotting was performed to monitor loading. Data are presented as mean ± SEM for three independent experiments. *p < 0.05; **p < 0.01 compared with control. e BV-2 cells were transfected with control or Nur77 siRNA for 48 h followed by treatment with 6-MP (50 μM) for 1 h (left) or 16 h (right). Total RNA and nuclear proteins were extracted. Expression levels of Nur77 mRNA and protein were analyzed by real-time RT-PCR (left) and western blotting (right), respectively. **p < 0.01 compared with 6-MP-treated control siRNA transfected cells. f, g After siRNA transfection, BV-2 cells were pretreated with 25 and 50 μM 6-MP for 16 h followed by treatment with LPS for another 2 h (f) or 6 h (g). Analyses of TNF-α mRNA expression and released TNF-α were performed as described in Fig. 1. Data are presented as mean ± SEM for three independent experiments. LPS-induced TNF-α production in control siRNA and Nur77 siRNA transfected cells were 25.1 ± 2.4 and 27.0 ± 3.5 ng/ml, respectively. *p < 0.05; **p < 0.01 compared with 6-MP-pretreated control siRNA transfected cells. h, i Nur77 contributes to 6-MP-mediated inhibition of NF-κB and TNF-α promoter transcriptional activities. After siRNA transfection, NF-κB (h) or TNF-α promoter (i) luciferase construct was transfected into BV-2 cells for 24 h. The transfected cells were pretreated with 50 μM 6-MP for 16 h followed by treatment with LPS for another 6 h. Luciferase activity from the non-stimulated cells transfected with control siRNA was arbitrarily set at 1.0 for the calculation of fold. Data are presented as mean ± SEM for four independent experiments. **p < 0.01 compared with 6-MP-pretreated control siRNA transfected cells