FIGURE 9.

Nrf2/p62 up-regulates the expression of Bcl-2/Bcl-xL, and cadmium increases binding activities of Nrf2 to the ARE regions of the Bcl-2/Bcl-xL promoter in the cadmium-transformed BEAS-2B cells. Normal or transformed BEAS-2B cells were transfected with siRNA specific to p62 or Nrf2. After overnight transfection, the cells were treated with or without 10 μm cadmium for an additional 24 h. The expression levels of Bcl-2/Bcl-xL or p62/Nrf2 were analyzed by Western blot (A). For the ChIP assay, consensus or putative ARE regions of the Bcl-xL (B) and Bcl-2 promoter (E) were discovered, respectively. Cadmium-transformed cells and non-transformed cells were treated with cadmium (10 μm) for 6 h. After isolating chromatin, it was immunoprecipitated with an anti-Nrf2 antibody or control mouse IgG. The Nrf2 binding to the Bcl-xL or Bcl-2 promoter was analyzed by a normal real-time PCR (C and F) or quantitative real-time PCR (D and G) with the specific primers for each ARE region of the promoter. The presented data follow the percentage input method and are normalized to each control. The results were expressed as the mean ± S.E. (error bars) relative to the control of triplicate experiments. *, p < 0.05; ***, p < 0.001 versus the vehicle control (ANOVA, Scheffe's test). β-Actin was used as a loading control. ND, not detectable.