TGF-β and Hypoxia/Reoxygenation Promote Radioresistance of A549 Lung Cancer Cells through Activation of Nrf2 and EGFR

Figure 2

Ligand-independent phosphorylation of EGFR was induced and positively regulated by Nrf2 following combined treatment with TGF-β and hypoxia. (a) Cells were treated with 1 ng/mL TGF-β and hypoxia for 2 h, followed by reoxygenation and further incubation for 22 h. Cells were then lysed, and phospho-EGFR, total EGFR, phospho-ErbB2, and total ErbB2 levels were determined by western blotting. GAPDH was used as a loading control. (b) Cells were pretreated with 10 μM AG1478 for 1 h, followed by combined treatment with 1 ng/mL TGF-β and hypoxia/reoxygenation. Cells were lysed 22 h later, and phospho-EGFR, Nrf2, and HO-1 levels were determined by western blotting. ((c) and (d)) Cells were transiently transfected with 10 nM of scrambled-siRNA (si-Scr), Nrf2-siRNA (c), or HO-1-siRNA (d) for 24 h. Cells were then treated with 1 ng/mL TGF-β and incubated with hypoxic medium for 2 h, followed by reoxygenation.

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