Fig. 2.

HIF-2α mediates hypoxic EGFR signaling activation. (A) HN6 cells exposed to hypoxia for the indicated times were analyzed by western blotting with primary antibodies against HIF-1α, HIF-2α and proteins related to the EGFR signaling pathway. Signals for phosphorylated EGFR (pEGFR) expression were obtained after short (s.e.) and long (l.e.) radiographic exposure. (B) Serum-starved HN6 and HN13 cells transfected with 10 nM of either non-targeted control (c), HIF-1α or HIF-2α siRNA were exposed to hypoxia for 6 h. Western blot analyses showed expression levels of total and phosphorylated EGFR. (C), whole cell lysates from serum-deprived parental HN6 cells (−), HN6 cells transfected with non-targeted control siRNA (c; 20 nM) or increasing doses of HIF-1α or HIF-2α siRNAs (5–20 nM) were obtained following 6 h of hypoxia and utilized for immunoblotting to assess phosphorylated status of EGFR, Akt, PLC-γ1 and S6. (D) TGF-α levels in conditioned medium from control siRNA or HIF-2α–siRNA-transfected HN6 cells cultured under normoxic or hypoxic conditions for 2 h were determined by enzyme-linked immunosorbent assay (top panel), *P< 0.01 versus normoxic control siRNA, HIF-2α siRNA as well as hypoxic HIF-2α siRNA cells. Western blotting confirmed HIF-2α knocked down and lack of EGFR phosphorylation in hypoxic HIF-2α siRNA-transfected cells (bottom panel). (E) Total RNA was isolated from HN6 cells transfected with either control siRNA or HIF-2α siRNA cultured under normoxic or hypoxic conditions for 2 h. Following reverse transcription of 2 μg total RNA, HIF-2α and TGF-α messenger RNA levels were detected by PCR. β-actin was used as an internal control for equal loading. (F) Total RNA was isolated from HN4 and HN6 cells cultured under normoxia or hypoxia for 6 h. Following reverse transcription of 2 μg total RNA, HIF–TGF-α and EGFR messenger RNA levels were detected by PCR. β-Actin was used as an internal control for equal loading.