Figure 5.

Inhibition of Nrf2 signaling activates apoptosis in PDCD4/Keap1 overexpressing cells. A. HO-1 mRNA levels were analyzed by qRT-PCR in two PDCD4-overexpressing cell lines. Expression levels were normalized against GAPDH mRNA. Results were derived from three independent experiments, and are plotted as fold change relative to the corresponding values in control cells (defined as 1.0). B. HO-1 protein level was determined by western blot analysis. C. PDCD4-overexpressing A549 cells were co-transfected with FLAG-tagged Keap1 and siRNA targeting Nrf2 for 24 h, and then cell proliferation was detected using a WST-8 assay. Mean ± SD shown for three independent experiments, and statistical significance was calculated by Student’s t-test (*, P<0.05; **, P<0.01). D. PDCD4-overexpressing A549 cells were transiently co-transfected with FLAG-tagged Keap1 and siRNA targeting Nrf2 for 24 h, and then analyzed by western blotting with antibodies against cleaved-caspase-3 and cleaved-PARP. E. PDCD4-overexpressing A549 cells were transfected with FLAG-tagged Keap1 and siRNA targeting Nrf2 for 24 h, and then immunostained with anti-cleaved caspase-3 (green) and counterstained with DAPI (blue). The stained cells were visualized on an Olympus confocal microscope. Scale bar; 50 µm.