Figure 3.

PDCD4 inhibits Nrf2 activity and nuclear localization. A. Western blots for Nrf2 and Keap1 in PDCD4-overexpressing cells. B. Two lung cancer cell lines (A549 and H460) stably expressing PDCD4 were co-transfected with pRL-TK Renilla and ARE (NRF2-luciferase) plasmids, and subjected to luciferase reporter activity assays. Mean ± SD shown for three independent experiments, and statistical significance was calculated by Student’s t-test (*, P<0.05; **, P<0.01). C. Subcellular localization of Nrf2 in A549 cells. A549 cells were transfected with the indicated amounts of PDCD4 WT plasmid for 24 h and then Nrf2 levels in the cytosolic and nuclear fractions of indicated cells were determined by western blotting. Lamin B was used as a nuclear protein marker, and β-actin as a loading control. D. PDCD4-overexpressing cell lines (A549) were immunostained with anti-Nrf2 antibody (green) and counterstained with DAPI (blue). E. Subcellular localization of Nrf2 in H460 cells. H460 cells were transfected with the indicated amounts of PDCD4 WT plasmid for 24 h and then Nrf2 levels in the cytosolic and nuclear fractions of indicated cells were determined by western blotting. Lamin B was used as a nuclear protein marker, and β-actin as a loading control. Scale bar; 20 µm. F. Two PDCD4-overexpressing cell lines (H460) were immunostained with anti-Nrf2 antibody (green) and counterstained with DAPI (blue).