C/EBPα enhances HBx-induced SOCS3 expression.

<p>(A) Schematic representation of the transcriptional response elements on the murine SOCS3 promoter, showing a distal SRE (−95 to −87), a proximal SRE (−71 to −64), an AP1 element (−105 to −99), a FoxO motif (−103 to 95), a GC-rich region (−58 to −52), and a putative C/EBPs binding site (−98 to −84). (B) The effects of HBx and C/EBPα on SOCS3 promoter activity. HepG2 cells were transfected with SOCS3 reporter construct, HBx, and C/EBPα as indicated for 48 hr, after which the ratio of reporter activities was utilized to measure the promoter activity of SOCS3. *<i>P</i><0.0001 compared with C/EBPα transfectants. (C) ChIP analysis on the SOCS3 promoter. HepG2 cells were transfected with expression vectors for HBx and C/EBPα as indicated and harvested for ChIP analysis with anti-C/EBPα antibody, or control IgG. The precipitated genomic fragments were amplified using primers flanking the C/EBPs binding sites on human SOCS3 promoters. Genomic DNA from total chromatin lysates was included as an input control. The precipitates were confirmed using quantitative real-time PCR. (D) The interaction between HBx and C/EBPα by coimmunoprecipitation. HA-tagged HBx was transiently expressed in HepG2 cells. Proteins in lysates were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies. (E) The induction of SOCS3 protein expression by HBx and C/EBPα. The C/EBPα protein was transiently expressed for 48 hr in Mock and stably HBx-transfected cells. Whole HepG2 cell extracts stably expressing HA-tagged HBx protein were immunoblotted with the SOCS3-specific antibody. (F), (G) The mRNA expression of SOCS3 by HBx and C/EBPα in RT-PCR (F) or quantitative real-time PCR (G). (H) The effect of C/EBPα knockdown on HBx-induced SOCS3 promoter activity. *<i>P</i><0.0001 compared with the indicated control. (I) The expression of SOCS3 proteins by HBx in the C/EBPα knockdown system. Whole cell lysates were immunoblotted with indicated antibodies. (J). (K) The expression of SOCS3 mRNA by HBx in the C/EBPα knockdown system. Total RNA was extracted from the cells, and the levels of SOCS3 mRNA were determined via RT-PCR (J) and quantitative real-time PCR (K).