Fig. 1

L-HDAg activates Twist promoter through binding with Smad3 on Smad binding elements (SBEs). a Huh7 cells were co-transfected with Twist promoter reporter pXP2-Twist with either L-HDAg- or S-HDAg-expressing plasmids of three genotypes. The pSV-β-galactosidase-expressing plasmid was co-transfected for monitoring transfection efficiencies. Luciferase activity was measured and normalized with β-galactosidase activity value. The fold change of luciferase activity relative to β-galactosidase activity were expressed as mean ± SD from three independent experiments. *: p < 0.05 (Student’s t-test), N.S.: no significant differences compared to Vector control. b Nucleotide sequence of proximal Twist promoter. + 1: principal transcription start site of Twist gene. Underlining: TATA box sequence. Notations below sequence: two potential Smad-binding sites (SBE site1 and site2) and mutated sequence of Smad-binding region. Underlining with solid arrow: position of forward or reverse primer for PCR amplification of ChIP. c Huh7 cells expressing L-HDAg or S-HDAg of three genotypes were chromatin IP’d with mouse anti-HDAg monoclonal Ab, anti-Smad3 Ab (positive control), or anti-mouse IgG (negative control). ChIP-enriched DNA samples were analyzed by PCR using Smad3 binding element (SBE)-specific primers with amplicon 196-bp. The original input was amplified by PCR with GAPDH promoter specific primers (Input-GAPDH, 166 bp). Densitometric analysis were indicated. Results shown are mean ± SD from five independent experiments. N.S.: no significant differences