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. 2015 Sep 12;6(33):34992–35003. doi: 10.18632/oncotarget.5252

Figure 1. Constitutive activation of EGFR sustains cell proliferation in the absence of ligands.

Figure 1

a. Serum starved PC3, DU145, A549 and HT-29 cells were tested for EGFR phosphorylation. b. PC3 and A549 cells grown in the absence of serum/ligands were tested for EGFR dimerization (crosslinked by DMP) and phosphorylation. Serum starved A549 and PC3 were treated with EGF +/− C225 at 2.5 ug/ml c. or AEE788 at 2.5 uM d. for 15 minutes and measured for EGFR phosphorylation, EGFR and Actin using Western blot. e. Schematic diagram of WT and extra cellular domain deleted EGFR (ΔECD-EGFR). f. Western blot analysis of protein samples for pEGFR and EGFR (Flag) isolated from HEK 293 cells transfected with vector alone, WT EGFR or ΔECD EGFR for 24 hours. g. Western blot analysis of protein samples for pAkt, Akt, pErk1/2, Erk1/2 and EGFR isolated from PC3 cells treated with EGF +/− AEE788 or EGF +/− C225 for 15 minutes similarly as cells used in c and d. h. A549 cells were treated with AEE788 or C225 for 5 days at the indicated concentrations and measured cell number. i. Colony formation assay on PC3 and A549 cells were treated with increasing concentrations of AEE788 or C225 as indicated in 6-well plate and colony formation was counted when the cells reached 80–90% confluence. Data are means of +/− SD of triplicates (Suppl Figure 1a and 1b). Asterisk indicates the statistical significance between treated group and DMSO (P-value ≤ 0.0001).