FIGURE 2.

ARE between nucleotides −3148 and −3140 on reverse strand of Bcl-2 gene promoter is essential for antioxidant induction of Bcl-2 gene expression. A, systematic representation and cloning strategy of mouse Bcl-2 gene promoter into pGL2B or pGL2P luciferase reporter vectors. Four putative ARE sequences of the Bcl-2 promoter, three on the reverse strand (AREr1, AREr2, and AREr3) and one on the sense strand (ARE-F1), are shown (upper left panel). Mouse Bcl-2 promoter deletions were separately cloned into pGL2B Luc reporter gene, and plasmids were separately transfected in Hepa-1 cells. Cells were treated with DMSO or 50 μm t-BHQ for 24 h, and luciferase activity was measured (right panels). B, Bcl-2-3.6WT (wild type) and Bcl-2–3.6MT (mutated AREr3) promoter plasmids were separately transfected in Hepa-1 cells and analyzed for luciferase gene expression. In the same experiment, AREr3 and mutant AREr3 were attached to SV40 basal promoter hooked to the luciferase reporter gene by cloning in vector pGL2P and transfected in Hepa-1 cells, and cells were treated with DMSO or t-BHQ (50 μm for 24 h) and analyzed for luciferase activity. Human NQO1 ARE-luciferase reporter plasmid was also transfected in Hepa-1 cells as a positive control for t-BHQ-mediated luciferase gene induction. All experiments were performed three times, and one set of data is presented. The data shown are mean ± S.D. of three independent transfection experiments (*, p < 0.05; **, p < 0.005; ***, p < 0.0001). V, vector control. Error bars indicate S.E. of triplicate samples.