FIGURE 10.

Transformation mediated by ER-retained EGFR Mut R1-6/L393H depends on PI3K signaling. A, whole cell lysates from serum-starved NIH 3T3 cells stably expressing WT EGFR or EGFR Mut R1-6/L393H (LH) and treated as indicated were immunoblotted with antibodies specific for phospho-Akt S473, total Akt, total EGFR, or actin (loading control). Before lysing the cells, EGF was added for 5 min at 100 ng/ml in the presence or absence of 10 μm LY294002 (LY). B, quantification of Akt phosphorylation in cells expressing WT EGFR or EGFR Mut R1-6/393H in the absence of EGF. Data were averaged from five independent experiments including those shown in A (error bars show S.E.; **, p < 0.01). C, the effect of the PI3K inhibitor LY294002 (5 μm) on the capacity of WT EGFR-, EGFR Mut R1-6-, or EGFR Mut R1-6/393H-expressing cells to grow in soft agar. The data shown represent the mean ± S.D. from three independent experiments, normalized to WT EGFR + EGF. **, p < 0.01; ****, p < 0.0001. D, the effect of mTOR inhibition on the capacity of WT EGFR-, EGFR Mut R1-6-, or EGFR Mut R1-6/L393H-expressing cells to grow in soft agar was assessed by adding rapamycin (Rapa, 25 nm) to soft agar cultures. The mean ± S.D. is from three independent experiments, normalized to WT EGFR + EGF. * p < 0.05; ***, p < 0.001.