FIG 7.

ORF2 stimulates coactivator enhancement of β-catenin-dependent transcription. (A) Neuro-2A cells were cotransfected with 0.1 μg of the Top-Flash luciferase reporter and 0.25 μg of β-catenin (S33Y). Where indicated, increasing concentrations of a plasmid that expresses HMGA1 (0.25 or 0.5 μg plasmid) were used to examine the effect that these coactivators have on TCF promoter activity. (B) Neuro-2A cells were cotransfected with 0.1 μg of the Top-Flash luciferase reporter and 0.25 μg of β-catenin (S33Y). Where indicated, increasing concentrations of a plasmid expressing CBP (0.25 or 0.5 μg plasmid) were used to examine the effect that these coactivators have on TCF promoter activity. (C) The effect of ORF2 on the ability of HMGA1 to stimulate β-catenin-dependent transcription was examined. Neuro-2A cells were transfected with the designated plasmids. (D) The effect of ORF2 on CBP to stimulate β-catenin-dependent transcription was examined in Neuro-2A cells. Neuro-2A cells were transfected with the designated plasmids. Dual-luciferase assays were performed at 48 h after transfection. The results for cells transfected with 0.1 μg of the Top-Flash luciferase reporter and the Renilla luciferase under the control of a minimal herpesvirus TK promoter (0.05 μg DNA) were normalized to a value of 1, and the fold activation was calculated for the other samples. For the studies whose results are presented in panels A to D, plasmid DNA concentrations for each transfection contained the same amount of DNA by adding empty expression plasmid (pCDNA3.1). Furthermore, Neuro-2A cells were transfected with the TransIT Neural transfection reagent (catalog number MIR2145; Mirus) according to the manufacturer's instructions. The results are averages from three independent experiments, and error bars denote standard errors. *, significant differences (P < 0.05) relative to the promoter activity of samples containing Top-Flash and β-catenin using a one-way analysis of variance and Fisher's least-significant-difference multiple-means comparison tests; ns, the differences between samples were not significant.