Figure 5: Pan-ErbB2 inhibition abolishes heterodimerization of ErbB2 irrespective of the presence of ligand.

(a,b) Inhibition of ligand-dependent and ligand-independent ErbB2 heterodimers. After preincubation with 100 nM of the anti-ErbB2 agents, ErbB2–ErbB3 dimerization was triggered with 1 nM heregulin-β1 (HRG) in BT474 cells (top) and ErbB2–EGFR dimerization with 4 nM epidermal growth factor (EGF) in SKBR3 cells (bottom). Receptors were crosslinked with reducible crosslinker DTSSP. (a) ErbB2 was immunoprecipitated by anti-ErbB2-C-terminal antibody, and the co-immunoprecipitated receptor was determined by immunoblot analysis. (b) Micrographs show HRG-induced morphogenesis (acini branching) after 12 days of co-treatment (top). Spheroid branching was quantified by three times scoring of 100 acini classified into three populations (bottom), depending on the number of buds per acinus. Scale bar, 50 μm. (c) Effect of the ErbB2-targeting agents on cell migration. SKBR3 cells were seeded to confluence, and scratch wounds of 500 μm were made in the monolayers. Cells were treated with the indicated agents (100 nM) and co-stimulated with 4 nM EGF or HRG. Left, representative micrographs were taken after 48 h. Right, the cell migration was expressed as per cent open area relative to the area at time point 0 h (*P value versus respective mock (EGF/HRG) or as drawn, two-sided, unpaired Welch’s t-test). Scale bar, 500 μm. (d) Effect of the ErbB2-targeting agents on cell invasion induced by HRG. SKBR3 cells pre-seeded on Matrigel in a Boyden-type invasion chamber were treated with the indicated agents (100 nM) and co-stimulated for 72 h with 1 nM HRG. Data are mean±s.e.m. (*P value versus mock (+HRG) or as drawn, Mann–Whitney test).