Figure 5

The CREB (pSer133CREB)-CBP cascade mediates HBV replication. RTD-PCR analysis of CBP gene expression after 10 µM Fsk (A) and 5 µM H-89 (B) treatment for 120 h. (C) Western blot analysis for CBP and phospho-CREB (Ser133) protein expression after 10 µM Fsk and 5 µM H-89 treatment for 120 h. Relative pixel intensities (right) were normalized to the GAPDH pixel intensity (DMSO = 1). The HBV cccDNA level was increased in the presence of 10 µM Fsk (120 h), as determined by RTD-PCR (D) and Southern blot analysis (E). The HBV cccDNA level was decreased in the presence of 5 µM H-89 (120 h), as determined by RTD-PCR (F) and Southern blot analysis (G). (H) CREB mRNA and protein levels detected by RTD-PCR and western blotting were decreased after transfection with CREB-siRNA. (I) RTD-PCR quantification of cccDNA accumulation in HepG2.2.15.7 and HepAD38 cells transfected with CREB-siRNA or control-siRNA. (J) Southern blot analysis confirming lower cccDNA levels in HepG2.2.15.7 and HepAD38 cells transfected with CREB-siRNA compared with control-siRNA. Results are expressed as the number of cccDNA copies per genomic μg (mean ± standard deviation) from three independent experiments. *P < 0.05 and **P < 0.01 versus the corresponding DMSO. Quantitative gene expression data represent the mean ± standard deviation of three independent experiments and were normalized to the expression levels of human GAPDH. *P < 0.05 and **P < 0.01 versus Control-siRNA (Control-siRNA = 1). Abbreviations: DM, DMSO; siCON, Control-siRNA; siCREB, CREB-siRNA.