Figure 4.

Knockdown of Nrf2 by RNA interference prevents cyclo(His-Pro)-mediated regulation of antioxidant gene expression in PC12 cells. Cells were transfected with 100 nM siRNA or 100 nM scRNA (scrambled RNA), as described in Experimental, followed by incubation with 50 μM cyclo(His-Pro) for 24 hrs. (A) Nrf2 mRNA abundance, as revealed by real-time PCR. (B) Nrf2 in nuclear extracts analysed by Western blotting, revealing a ∼50% decrease in Nrf2 protein content. Results are given as mean ± S.D. values for n = 3 independent experiments. *P< 0.05 versus control. #P < 0.05 versus siRNA values. (C) Real-time PCR analyses showing expression of Nrf2 downstream genes. Changes in gene expression were normalized to p-actin expression and presented as 2^−ΔΔCT. Relative mRNA abundance of each gene in untreated cells was assumed to be 1.0 (control). (D) Cell viability (MTT reduction), ROS, NO and glutathione (GSH) were assessed in Nrf2-silenced cells treated with 50 μM cyclo(His-Pro) for 24 hrs prior to H₂O₂ treatment. Control values (100%) are indicated in the Figure 2 legend. Results are given as mean ± S.D. values for n = 3 independent experiments. *P<0.05 versus CHP+H₂O₂- Nrf2scRNA.