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

Figure 4

Generation of ROS by TGF-β and hypoxia regulated Nrf2 and EGFR activation. ((a) and (b)) A549 cells were treated with TGF-β and hypoxia for 2 h followed by reoxygenation for 1 (a) or 22 h (b). After reoxygenation, ROS levels were measured using a fluorometer (a) or FACS analysis (b). Cells were pretreated with 1 mM N-acetyl-L-cysteine (NAC) for 1 h before TGF-β and hypoxia/reoxygenation. Data are representative of at least three independent experiments. versus the corresponding value for control ROS. (c) Nuclear extracts or whole cells were lysed after cotreatment with TGF-β and hypoxia for 2 h, followed by reoxygenation for 6 h. Nrf2, HO-1, and phospho-EGFR levels were then determined by western blotting. Histone H3 and GAPDH were used as loading controls. (d) Cells were treated as described in (c), and whole cell lysates were analyzed by western blotting for caveolin-1, NOX1, NOX2, and NOX4.

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