Fig. 3

CDCP1 and EGFR cooperate to induce cell detachment from the substratum and to disrupt adherens junctions. a Immunoblot analysis of T47D cell lines stably expressing the indicated combinations of EGFR, CDCP1, and the Y734F mutant CDCP1. Coexpression of wild-type CDCP1 and EGFR results in a tyrosine-phosphorylated band that comigrates with EGFR. Similar results were observed whether the cells are plated on tissue culture plastic or type I collagen. b Coexpression of CDCP1 and EGFR result in enhanced EGF-dependent EGFR phosphorylation on Tyr⁸⁴⁵. This effect was blunted if wild-type CDCP1 was replaced with the Y734F mutant. c Treatment with DDA NSC624205 (20 μM) for 24 hours induces an electrophoretic mobility shift of EGFR, but not CDCP1, in the T47D cell background. d T47D cells stably expressing EGFR and CDCP1 were treated for 24 hours with vehicle or 20 μM DDA NSC333839 and stimulated for 20 minutes with or without 10 ng/ml EGF and analyzed by immunoblot. e The indicated cell lines were plated onto tissue culture plastic and treated with or without 10 ng/ml EGF for 24 hours and photographed. f Cells grown and treated as in Fig. 3b were gently pipetted with the culture medium and the detached suspension cells, and the cells remaining attached to the plates were counted. Results were plotted as the fraction of cells that were detached in triplicate determinations. Error bars represent standard deviation (SD). g Photomicrographs of the indicated cell lines treated as in Fig. 3e, but grown on the surface of collagen I gels. Note that EGFR and CDCP1 coexpression and EGF stimulation cooperate to induce suspension growth