Figure 4: DBT induces the estrogenic effects in cultured MG-63 cells. (a) Three repeats of estrogen responsive elements (ERE: 5′-GGT CAC AGT GAC C-3′) was subcloned into a promoter-reporter vector that has a down stream reporter of firefly luciferase gene, namely as pERE-Luc. (b) Cultured MG-63 cells that stably transfected with pERE-Luc, or transiently transfected with pMEF2-Luc or pE-box-Luc were treated with different concentration of β-estradiol (E2; from 10 nM to 1 μM) for 48 h to determine the transcriptional activity of pERE-Luc by luciferase assay. (c) Total RNAs were extracted from MG-63 cells to determine the presence of estrogen receptor α (ERα; 510 bp) and β (ERβ; 259 bp) by RT-PCR analysis. No RT indicated the absence of contamination by genomic DNA, and ERα and ERβ cDNAs served as positive control for PCR. Representative images were shown, n = 5. (d) Cultures were treated with extracts (1 mg mL−1) derived from DBT, RA, RAS and RA + RAS for 48 h to determine the luciferase activity as in (b). (e) A dose-response curve of DBT was performed as in (b). Values of the promoter-driven luciferase activities are expressed in percentage of increase as compared with control cultures (without herbal extract), and in mean ± SEM, where n = 5, each with triplicate samples. **P < .01.