FIGURE 2.

EGFRvIII-expressing GBM cells generate increased levels of suPAR. A, parental U373MG cells and U373MG cells that overexpress WT-EGFR (EGFR) or express EGFRvIII (vIII) were serum-starved for 18 h. The cells were then treated with 10 ng/ml EGF (+) or with vehicle (−) for an additional 18 h. CM was recovered, concentrated 50×, and subjected to immunoblot analysis to detect suPAR. The cells from which CM was recovered also were subjected to immunoblot analysis to detect tubulin, as a control to ensure that CM was generated by an equivalent load of cells. Mean relative signal intensities are shown below the blots (n = 3). In each case, the signal intensity is standardized against that observed in the absence of EGF. B, U87MG cells were treated as described in A. CM was subjected to immunoblot analysis to detect suPAR. Cell extracts were analyzed to detect tubulin. C, U373MG cells that express EGFRvIII under the control of a doxycycline (DOX)-repressible promoter were treated (+) or untreated (−) with 1 μg/ml doxycycline for 3 days and subsequently serum-starved for 24 h in the presence or absence of doxycycline. Immunoblot analysis was performed to detect suPAR in CM and uPAR in cell extracts. EGFR and actin in cell extracts also were assessed. D, U87MG cells were cultured in serum-free medium with GST-RAP or GST (150 nm) for 24 or 48 h. CM and cell extracts were subjected to immunoblot analysis to detect uPAR and actin.