Wild-type EGFR increases proliferation rate in complete medium.

<p>(A,B) AD293 cells overexpress EGFRvIII, EGFRwt or both receptor variants. Cells were cultured in complete medium, collected and lysed. Western blots show levels of protein expression of total EGFR (sc-03 antibody), phospho-EGFR (tyr1173 antibody), phospho-AKT, AKT, phospho-STAT5 and Actin in four different cell lines (A). Real-time PCR was used to evaluate relative expression of total EGFR (wt and vIII) against <i>HPRT1</i> housekeeping gene in AD293 cell lines (B). <i>Statistical significance calculated against a value of specific gene in AD293par</i>, <i>ns</i>: <i>P>0</i>.<i>05; ***</i>: <i>P<0</i>.<i>001</i>. (C) EGFRwt expressing cells have the highest proliferation rate. Cells were seeded in complete medium and after 6 hours were moved to Nikon BioStation CT for live observation. Student’s t-test: ns: P>0.05; ***: P<0.001. (D) EGFRvIII expressing cells have constitutive expression of cyclin D1 independently on EGF treatment. Cells were serum starved for 24 hours, then supplemented with DMSO (control), EGF (50 ng/ml) or EGF/erlotinib (15 μM). After 24 hours cells were lysed and real-time RT-PCR was conducted. <i>Statistical significance calculated against a value for AD293par from the same group</i>, <i>ns</i>: <i>P>0</i>.<i>05; *</i>: <i>P<0</i>.<i>05; ***</i>: <i>P<0</i>.<i>001</i>. (E, F) EGFRwt has a peak activity after 1 hour of EGF treatment. Cells were serum starved for 24 hour and then serum free medium with EGF (20 ng/ml) was added. After specified time cells were lysed and blotted for total EGFR, phospho-EGFR and Actin (E). Bands intensity was calculated with ImageJ software (F). (G) AD293 cells overexpressing EGFRvIII have constitutively activated AKT and STAT5, while EGFRwt induces such activation after EGF treatment. Cells were serum starved for 24 hour and then serum free medium with EGF (20 ng/ml) or EGF/erlotinib (15 μM) was added. After 1 hour cells were lysed and blotted for phospho-EGFR, phospho-AKT, AKT, phospho-STAT5 and Actin.