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. 2007 Aug 1;104(32):13092–13097. doi: 10.1073/pnas.0702387104

Fig. 2.

Fig. 2.

HIF2α activation results in induction of EGFR protein levels. (A) Western blot analysis of EGFR, HIFα, and GLUT levels in U87MG cells infected with adenovirus expressing flag-tagged GFP or a dominant-negative form of HIF (dnHIF). Cells were grown as 3D spheroids for 3 days. Control cells were grown in 2D culture for the same period. (B) Western blot analysis of EGFR and HIFα levels in U87MG and MDA-MB-231 cells infected with GFP or dnHIF for 24 h. before exposure to normoxia or hypoxia for an additional 24 h. (C and D) Western blot (C) and RT-PCR (D) analysis of EGFR levels in U87MG cells infected to express GFP or HIFα variants (HIF1α and HIF2α) for 72 h in normoxia. (E) Western blot analysis of EGFR and HIFα levels in U87MG transiently transfected with siRNA (50 nM) targeting HIF1α (siHIF1α), HIF2α (siHIF2α), or a scramble sequence (siCont) for 48 h before exposure to hypoxia for an additional 24 h. (F) Western blot analysis of EGFR and HIF2α levels in U87MG pretreated with 10 nM rapamycin (Rap), or DMSO as a vehicle control, for 1 h and then exposed to normoxia or hypoxia for an additional 16 h. The phosphorylation status of the S6 ribosomal protein (ph-S6) served as a control for drug activity. Actin served as a loading control in A–F.