FIGURE 8.

Overexpression of INrf2 leads to degradation of PGAM5-Bcl-xL and activation of pro-apoptotic proteins. A, INrf2-293 cells transfected with PGAM5-V5 plasmid and treated with tetracycline for 24 h and Hepa-1 cells co-transfected with FLAG-INrf2 and PGAM5L-V5 plasmids for 24 h were treated with the indicated concentrations of etoposide for an additional 24 h. Cells were lysed and immunoblotted (left and right). B, Hepa-1 cells were transfected with pcDNA vector or FLAG-INrf2 and treated with etoposide (20 μm) for 36 h. Cells were harvested, and mitochondria were isolated, and mitochondrial and cytosolic lysates were immunoblotted with anti-cytochrome c, CoxIV, and actin antibodies. C, Hepa-1 cells were transfected with pcDNA vector or FLAG-INrf2 and treated with etoposide (20 μm) as indicated for 36 h. Similarly, INrf2-293-cells were treated with tetracycline followed by treatment with 1 μm etoposide for an additional 36 h. Cells were lysed, and 20 μg of cell lysates were mixed with Caspase Glo 3/7 substrate (Promega), and caspase-3/7 activity was measured and plotted (top and bottom). Data are mean ± S.D. from three independent experiments. D, 60 μg of INrf2-overexpressed or etoposide-treated cell lysates of Hepa-1 cells and INrf2-293 cells from C were immunoblotted with anti-caspase-3, anti-FLAG, and anti-actin antibodies (left and right).