Involvement of AP-1 in MMP-9 expression with JEV infection of RBA cells. (A, B) Cells were pretreated with tanshinone for 1 h or transfected with siRNA of scrambled (scrb), c-Jun, or c-Fos, and then infected with JEV for 16 h. The conditioned media were used to determine MMP-9 expression by gelatin zymography. Cell lysates were analyzed by western blot using an anti-c-Jun, anti-c-Fos, or anti-GAPDH antibody. (C) Cells were pretreated with tanshinone for 1 h, and then incubated with JEV for 6 h. RNA samples were analyzed by RT-PCR to assess the levels of MMP-9 mRNA expression. (D, E) Cells were transiently transfected with MMP-9-luc reporter gene, pretreated with tanshinone (10 μM) for 1 h, and then incubated with JEV for 6 h. In addition, cells were transfected with wild-type MMP-9 promoter and AP-1-mutated MMP-9 promoter, and then incubated with JEV (moi = 1) for 6 h. MMP-9 promoter activity was determined in the cell lysates. Data are expressed as mean ± S.E.M. for five independent experiments. *P < 0.05; #P < 0.01, as compared with the cells exposed to JEV alone (A, D). #P < 0.01, as compared with cells transfected with wild-type MMP-9 promoter stimulated by JEV (E).
Yang et al. Journal of Neuroinflammation 2012 9:12 doi:10.1186/1742-2094-9-12