Figure 6
Role of p21Cip1 in the antioxidative response, which decreases ROS generation and apoptosis in Jdp2- KO GCPs. (a) Increased protein interaction between endogenous p21Cip1 and Nrf2 in Jdp2-KO GCPs. Cell lysates (120 μg) from WT and Jdp2-KO GCPs were immunoprecipitated with anti-p21Cip1 (left panel) or anti-Nrf2 (right panel) antibodies, and the bound proteins were blotted with anti-Nrf2 (left panel) or anti-p21Cip1 (right panel) antibodies as described in Materials and Methods. IgG was used as a negative control. The relative intensity was calculated relative to β-actin. (b–g) Effects of siRNA against p21Cip1on the Slc7a11 promoter (b) and ARE (c) activities, cystine uptake (d), intracellular level of GSH (e), ROS production (f) and apoptosis (g). WT and Jdp2-KO GCPs were transfected with 30 pmol of p21Cip1 siRNA (siRNA 1 or 3), Slc7a11 siRNA (#1, #2, and #3), or control scrambled siRNA for 24 h, and the cells were harvested and analyzed as described in Materials and Methods. Values represent the mean ± SEM (n = 3). *p < 0.05. (h) A schematic representation of the interactions between Jdp2, sMaf, Nrf2, and p21Cip1 on the ARE of the Scl7a11 promoter in WT and Jdp2-KO GCPs, which regulates ROS-induced apoptosis of GCPs. In the absence of Jdp2, the endogenous interactions between Nrf2 and p21Cip1 at the ARE of Slc7a11 promoter increased, which increased cystine uptake, intracellular cysteine level, and the GSH/GSSG ratio and led to decreases in ROS and apoptosis in GCPs.
