After transfection, cells were treated with MMP-9 inducer 12-O-tetradecanoylphorbol-13-acetate (TPA), and then luciferase activities RAD001 in vivo were

determined. HBx or AIB1 alone can induce MMP-9 promoter activity to 5- or 3-fold, whereas the coexpression of HBx and AIB1 dramatically increased MMP-9 promoter activity to more than 10-fold (Fig. 6C). These results suggest that HBx can cooperate with AIB1 to increase MMP-9 promoter activity. To determine whether the cooperative effect of HBx and AIB1 on MMP-9 promoter activity is simply the result of elevated AIB1 protein levels, MMP-9 promoter/reporter assays were performed after HBx was transfected along with WT AIB1, AIB1-S505A, and AIB1-S509A, respectively. Similar to WT AIB1, HBx could cooperate with AIB1-S505A and AIB1-S509A to promote MMP-9 promoter activity (Fig. 6D). These results exclude the possibility that the cooperative effect of HBx and AIB1 on MMP-9 promoter activity is solely dependent on the elevation of AIB1 protein levels, because the protein levels of

AIB1-S505A and AIB1-S509A were almost not affected by HBx (Fig. 4D). We previously showed that AIB1 could be recruited to the MMP-9 promoter.17 Therefore, it is possible that HBx and AIB1 can co-occupy the MMP-9 promoter if these two proteins are stably associated. To test this hypothesis, ChIP assays were performed. Results DNA Damage inhibitor showed that HBx Tacrolimus (FK506) was recruited to the MMP-9 promoter, and that recruitment was enhanced after the overexpression of AIB1 (Fig. 6E, lane 6 versus lane 5); similarly, AIB1 was recruited to the MMP-9 promoter, and recruitment was enhanced by HBx (Fig. 6F, lane 6 versus lane 5). These results indicate that both HBx and AIB1 are recruited to the MMP-9 promoter to cooperatively enhance MMP-9 promoter activity. To further confirm the cooperative role of HBx and AIB1 in MMP-9 expression, HepG2 cells, which highly express AIB1 (AIB1WT), but do not contain the HBx gene, were stably transfected with AIB1-knockdown (AIB1KD) plasmids to establish AIB1KD/HBx−

cell lines, HBx expression plasmids to establish AIB1WT/HBx+ cell lines, both AIB1-knockdown plasmids and HBx expression plasmids to establish AIB1KD/HBx+ cell lines, and control plasmids to establish AIB1WT/HBx− cell lines, respectively. The expression of AIB1 protein was dramatically reduced in AIB1KD/HBx− cells, compared to AIB1WT/HBx− cells; ectopic expression of HBx significantly up-regulated AIB1 protein levels in both AIB1KD/HBx+ and AIB1WT/HBx+ cells, compared to AIB1KD/HBx− and AIB1WT/HBx− cells, respectively (Fig. 7A). The protein levels of HBx in AIB1KD/HBx+ and AIB1WT/HBx+ cells were comparable to that in human HBx-positive HCC tissues (Supporting Fig. 1).