Fig. 3

Activation of Twist promoter by L-HDAg was strongly reduced by disruption of SBEs, C211S mutation of L-HDAg, or Smad3 knockdown. a Wild type Twist promoter reporter (pXP2-Twist) or SBEs mutated reporter (mt-pXP2-Twist) were co-transfected into Huh7 cells with plasmids encoding L-HDAg, C211S mutant of L-HDAg (L-C211S), S-HDAg, or pcDNA control vector. The fold change of luciferase activity relative to β-galactosidase activity were shown as mean ± SD from three independent experiments. *: p < 0.05; **p < 0.01 (Student’s t-test). b Effect of Smad3 knockdown on Twist promoter activity, by luciferase assay. Plasmids expressing Smad3-targeting shRNA (shSmad3) and non-targeting control (shLuc) were transiently transfected with pXP2-Twist and L-HDAg, L-C211S, S-HDAg, or pcDNA control plasmids. Transfectants lysate were analyzed by luciferase assay (left panel) or immunoblotting (right panel) with anti-Smad3 Ab, antiserum from delta antigen hepatitis patients, or anti-Hsp70 Ab as internal control. *: p < 0.05 (compared with vector control in shLuc group). c Prenylation-deficient mutants of L-HDAg (C211S) from all three genotypes lost the ability to activate Twist promoter. The pXP2-Twist reporter was co-transfected with L-HDAg, L-C211S, S-HDAg, or pcDNA3 control plasmids, and transfectants were analyzed by luciferase assay