FIGURE 5.

siRNA inhibition of Nrf2 decreases t-BHQ-inducible expression of Bcl-2. A, Western analysis. Hepa-1 cells were transfected with control or 25, 50, or 75 nm Nrf2 siRNA. Forty-eight hours after transfection, cells were harvested, lysed, and immunoblotted with anti-Nrf2, anti-Bcl-2, anti-NQO1, and anti-actin antibodies. B, real time PCR analysis. Hepa-1 cells were transfected with control or Nrf2 siRNA. Twenty-four hours after siRNA transfection, cells were harvested, and total RNA was extracted and converted to cDNA. cDNA (50 ng) was analyzed for mRNA levels using Bcl-2 and NQO1 primers and probes. C, real time PCR analysis. Hepa-1 cells were transfected with control or Nrf2 siRNA (75 nm). Twenty-four hours after siRNA transfection, cells were treated with t-BHQ for an additional 16 h, cells were harvested, and total RNA was extracted. The mRNA levels of Bcl-2, NQO1, and Nrf2 were quantified by real time PCR. D, reporter analysis. Hepa-1 cells were transfected with control or Nrf2 siRNA. Twenty-four hours after transfection, cells were transfected with wild type or mutant Bcl-2-3.6 (left panels) or with AREr3 or mutant AREr3 plasmids (right panels), incubated with DMSO or t-BHQ (50 μm) for 24 h, and analyzed for luciferase activity. All experiments were performed three times. The data shown are mean ± S.D. of three independent transfection experiments (*, p < 0.05; **, p < 0.005; ***, p < 0.0001). Error bars indicate S.E. of triplicate samples.