Figure 3

Regulation of c-Myc-mediated Hsp90α promoter transactivation by HBx. (A) Schematic illustration of the genomic region encompassing the 5' flanking region of the human the Hsp90α promoter and the reporter constructs used. (B) Cells were cotransfected with or without 1 μg of pcDNA3 or pcDNA3-X plasmid, 2 μg of Hsp90α promoter-luciferase reporter construct, and and 2 μg of β-galactosidase reporter plasmid by the LipofecAMINE method. Cells were cultured in 10% FBS medium for 24 h. Luciferase activity and β-galactosidase activity were assayed by using the luciferase and β-galactosidase enzyme assay system. Luciferase activity was normalized with the β-galactosidase activity in cell lysates. Error bars indicate standard deviations (SD) obtained from three different experiments prepared in triplicate. (C) The nuclear extract isolated from HepG2-pcDNA3 (lane 1-3) and HepG2-pcDNA3-X (lanes 4-6) cells incubated with [γ^-32P]ATP-labeled oligonucleotides corresponding to the wt c-Myc binding site of the Hsp90α promoter. Competition was performed using unlabeled wt oligo or mutant oligo. Each sample was electrophoresed in a 4% nondenaturing polyacrylamide gel in 0.5 × Tris-borate EDTA buffer at 250 V for 20 min. The gel was dried and subjected to autoradiography.