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. 2016 Mar 21;5(3):e211. doi: 10.1038/oncsis.2016.25

Figure 3.

Figure 3

Combination of C3E10 with either trastuzumab or pertuzumab synergistically inhibits ErbB2 heterodimerization and signaling. (a) Competitive binding assay. 3E10, trastuzumab and pertuzumab were evaluated for their ability to compete with Alexa Fluor 488-labeled 3E10, Alexa Fluor 488-labeled trastuzumab or Alexa Fluor 488-labeled pertuzumab for binding to ErbB2-overexpressing BT-474 cells. (b) Coimmunoprecipitation assay comparing the ability of control IgG (10 μg/ml), trastuzumab (10 μg/ml), C3E10 (10 μg/ml) and trastuzumab plus C3E10 (5 μg/ml each) to disrupt ligand-independent ErbB2/ErbB3 heterodimer formation in BT-474 cells. (c) Coimmunoprecipitation assay detecting EGF-induced ErbB2/EGFR and HRG-induced ErbB2/ErbB3 heterodimerization in BT-474 cells pretreated with control IgG (10 μg/ml), pertuzumab (10 μg/ml), C3E10 (10 μg/ml) or pertuzumab plus C3E10 (5 μg/ml each). (d) Immunoblots examining ErbB2 signaling in BT-474 cells upon treatment with control IgG (10 μg/ml), trastuzumab (10 μg/ml), C3E10 (10 μg/ml) or trastuzumab plus C3E10 (5 μg/ml each) in the absence of ErbB ligand. (e) Immunoblots assessing the effects of control IgG (10 μg/ml), pertuzumab (10 μg/ml), C3E10 (10 μg/ml) or pertuzumab plus C3E10 (5 μg/ml each) pretreatment on EGF- or HRG-activated ErbB2 signaling in BT-474 cells. (f) MTS assay comparing the effects of control IgG (10 μg/ml), trastuzumab (10 μg/ml), C3E10 (10 μg/ml) and trastuzumab plus C3E10 (5 μg/ml each) on BT-474 cell proliferation in the absence of ErbB ligand. Results are shown as percentage of control cell proliferation. Error bars, s.d. *P<0.05, **P<0.001. (g) MTS assay assessing the effects of control IgG (10 μg/ml), pertuzumab (10 μg/ml), C3E10 (10 μg/ml) and pertuzumab plus C3E10 (5 μg/ml each) on BT-474 cell proliferation in the presence of HRG or EGF. Results are shown as percentage of control cell proliferation. Error bars, s.d. *P<0.05.