Involvement of TCF7L2 in up-regulation of core promoter activity. (a) Transcription-related proteins from database search (HNF1α, SRY, TCF7L2, SP1, FOXM1 and KLF5) and PUF60 were synthesized in vitro and used for the gel shift assay with an end-labeled oligonucleotide probe (nt 1689–1726). (b) Interaction of PUF60 with TCF7L2 in cells was tested. Cells transfected with pcDNA-HA-PUF60 or -PUF60-D2 and pcDNA-F-TCF7L2 or -HNF4α plasmids were lysed at day 2 pt and subjected to immunoprecipitation (IP) with anti-HA antibody. Resulting precipitates and whole cell lysates were examined by immunoblotting using anti-FLAG, anti-HA or anti-GAPDH antibody. (c) ChIP assay was performed to determine recruitment of PUF60 to the core promoter in cells. After 2 days with or without knockdown of TCF7L2, cells were transfected with pcDNA-F-PUF60 or empty vector (EV). Further 2 days later, cell lysates were immunoprecipitated with anti-FLAG antibody, and HBV DNA in the precipitates was measured by qPCR. FLAG-PUF60 and GAPDH in the precipitates were detected by immunoblotting. (d) Effect of TCF7L2 expression on ENII/BCP activity was determined by the reporter assay. Cells were transfected with pcDNA-F-PUF60 or -TCF7L2 or both and pGLHBp1627/1817. At 24 h pt, Renilla luciferase activities in cells were measured. (e) Effect of TCF7L2 over-expression with or without PUF60 on 3.5 kb RNA expression was determined. Cells were transfected with pcDNA-F-PUF60 or -TCF7L2 or both and pUC-HB-Ce. At day 2 or 4 pt, total RNA was extracted and HBV 3.5 kb RNA level was assessed by RT-qPCR. Results were normalized to that of β-actin mRNA. (f) Effect of FBP over-expression with or without PUF60 on core promoter and ENII/BCP activities was assessed. Cells were transfected with pcDNA-HA-FBP or -F-PUF60 or both and pGLHBp900/1817 or pGLHBp1627/1817. Luciferase activities in cell lysates were measured at day 2 pt. (d)–(f) The values in cells transfected with EV are set to 1. Results are presented as means ± SD from at least three independent samples. Statistical differences compared with the negative control (EV only) are shown. *p < 0.05, **p < 0.01, Student’s t test. Full-length blots in (b) and (c) are presented in Supplementary Figures S17 and S18, respectively.