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Fig. 1 | Journal of Biomedical Science

Fig. 1

From: Hepatitis B virus X protein (HBx) enhances centrosomal P4.1-associated protein (CPAP) expression to promote hepatocarcinogenesis

Fig. 1

CREB is crucial for HBx to associate with CPAP promoter and to enhance the CPAP promoter activity. a (i) Huh7 or Hep3B cells with GFP or GFP-HBx expression were collected to detect the expression of CPAP and GFP-HBx by Western blot analysis. The mRNA level (ii) and promoter activity (iii) of CPAP in HepG2, HepG2X, Hep3B and Hep3BX cells were determined by RT-qPCR (for CPAP mRNA) or RT-PCR (for HBx mRNA) and reporter assay, respectively. The expression levels of endogenous CPAP are indicated as ratio (CPAP/Tubulin). The mean ± SD was obtained from triplex Q-PCR, and three independent experiments were performed. Error bars of reporter assay represent the mean ± SD of three independent experiments, each performed in triplicate. b The expressions of CPAP (left) and HBV surface antigen (HBs) (right) were determined by Q-PCR or RT-PCR and Western blot analysis in HBV-infected (+) or no infected (−) HepG2-NTCP-C4 cells [14]. c (i) A schematic representation of the CPAP proximal promoter (pGL2-CPAP promoter/0.5 kb) is shown. Two potential CREB binding sites (CBSs) are showed in black ovals (WT), and the promoter with either one or both CBSs mutations is presented by gray color (M1, M2, or M1 + M2). (ii) CPAP promoter activity was examined in Huh7 cells with GFP-CREB or/and HA-HBx expression by reporter assay. d pGL2-CPAP/WT, M1, M2 or M1 + 2 was transiently transfected into Huh7 cells with GFP-CREB (left) or GFP-HBx (right), and then, the luciferase activity was analyzed. Error bars represent the mean ± SD of three independent experiments, each performed in triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001; N.S., no significance. e (Left) Primers used in chromatin immunoprecipitation (ChIP) and re-ChIP assay of the CPAP promoter are shown. The ChIP (middle) and re-ChIP (right) assay were performed in GFP-HBx- and HA-CREB-expressing Huh7 cells by anti-GFP, anti-HA antibody and normal serum control. f pGL2-CPAP/WT or M1 and HA-CREB (left) or GFP-HBx (right) were co-transfected into Huh7 cells, and then analyzed by ChIP assay using anti-HA or anti-GFP antibody as described in (e). WC: water control

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