To further investigate the possible effect of KLF15 on HBV gene expression from the HBV genome, we cotransfected pKLF15 or its control vector with pHBV1.3D, which contains the 1.3-mer HBV genome, into HepG2 and Huh7 cells. Our results showed that the coexpression of KLF15 led to a seven-fold increase of the HBsAg level in the culture medium of HepG2 cells (Fig. 3A). Such an increase in HBsAg production was even more prominent in Huh7 cells, which was up to nearly 20-fold (Fig. 3B). Similarly, KLF15 also increased the core protein expression level in HepG2 cells (Fig. 3C). When the culture

medium and cell lysates from transfectants were analyzed for encapsidated HBV DNA by RT-PCR, we found that the coexpression of KLF15 increased the extracellular encapsidated HBV DNA level by approximately two-fold and also slightly increased the intracellular encapsidated Y-27632 datasheet HBV DNA level (Fig. 3D). Taken together, our results indicated that, in the context of the HBV genome, KLF15

could enhance RGFP966 price the expression of HBsAg and the core protein, as well as HBV DNA replication. To determine whether endogenous KLF15 would also regulate HBV gene expression, we used siRNA to reduce the expression of endogenous KLF15. As shown in Fig. 4A, the transfection of KLF15 siRNA into Huh7 cells resulted in an approximately 70% reduction of the KLF15 messenger RNA (mRNA) level. This reduction of KLF15 expression led to an approximately 50% reduction in HBsAg expression from the HBV genome (Fig. 4B). Consistent with this result, KLF15 siRNA also reduced the luciferase activities of the HBV core promoter and the surface promoter by approximately 50% and 30%, respectively for (Fig. 4C and 4D). Thus, the results shown in Fig. 4 indicated that endogenous KLF15 also positively regulates HBV surface and core promoters. To characterize the mechanism by which KLF15 binds to core and surface promoters, the FLAG-tagged KLF15 protein

was expressed in 293T cells and purified with an anti-FLAG affinity gel (Fig. 5A). Although the crude cell lysates also contained Sp1 and NF-Y that are known to interact with the S promoter (Fig. 5A, lane 1), these two protein factors were found only in the unbound fraction (Fig. 5A, lane 2) and not in the affinity-purified KLF15 fraction (Fig. 5A, lane 3), indicating the specificity of this purification. Using EMSAs, we showed that rKLF15 was able to bind to labeled core promoter probe CP35 (Fig. 5B). KLF15-DNA binding was specific, as the addition of 100-fold nonlabeled CP35 disrupted the protein-DNA complex (Fig. 5B, lane 2). It has been very well demonstrated that a functional KLF15 binding site is present in the CLCK1 gene promoter.