Fig. 6

EpAb2-6 inhibits EpCAM and HGFR signaling and promotes active β-catenin and Snail protein degradation via activating GSK3β. A HCT116 cells were treated with 10 μg/ml control IgG (normal mouse IgG, NMIgG) or EpAb2-6 for 16 h, followed by treatment with EpEX-His (50 nM) for 15 min. Levels of phosphorylated HGFR, AKT, FAK, GSK3β, ERK, ADAM17, and presenilin 2 were examined by Western blotting. B HCT116 cells were treated with 10 μg/ml control IgG or EpAb2-6 for 16 h, followed by treatment without or with HGF (0.5 nM) for 15 min. Levels of phosphorylated HGFR, AKT, and ERK were examined by Western blotting. HCT116 cells were treated with EpAb2-6 (10 μg/ml) and HGF (0.5 nM). C Cells pre-treated with 5 µg/ml mitomycin-C for 4 h and then treated with IgG or EpAb2-6 and/or HGF. Cell migration was examined by the wound healing assay at the indicated times. D Cell invasion was assessed by a Transwell assay with matrigel after 24 h. E HCT116 cells were treated with NMIgG or EpAb2-6 (20 μg/ml) for 24 h and then immunoprecipitated with anti-EpCAM (IP: EpCAM) or anti-HGFR (IP: HGFR) antibodies, followed by Western blotting. F HCT116 cells were treated with NMIgG, MT201, EpAb2-6 or humanized EpAb2-6 (hEpAb2-6) (20 μg/ml) for 24 h. Representative images of TIRF-FRET experiments showing energy transfer from HGFR to EpEX in HCT116 cells. HGFR as donor channel (AF488) and EpEX as acceptor channel (AF568). Negative control (NC): Donor HGFR-AF488 and acceptor normal IgG-AF568. G EpEX-His (2 μg/ml) co-treated with 1 μg IgG or EpAb2-6 was added to HGFR-Fc-coated (1 μg/ml) ELISA plates and detected by TMB colorimetric peroxidase assay. H β-catenin and Snail protein levels were detected by Western blotting in HCT116 cells treated with NMIgG or EpAb2-6 for 24 h. I HCT116 cells were treated with 10 μM MG132 and EpAb2-6 for 6 h before cell collection and subsequent Western blotting. J Stability of Snail protein in HCT116 cells treated with NMIgG or EpAb2-6. Cells were treated with cyclohexamide (CHX) 100 μg/ml at the indicated intervals and subjected to Western blotting. Bottom graph shows quantification of Snail half-life in indicated groups. K Invasion assays were performed using HCT116 cells expressing Snail-WT, -2SA, or -4SA plasmids, with or without EpAb2-6 treatment. L Protein expression was analyzed by Western blotting in HCT116 cells after treatment with EpAb2-6 and 2 μM GSK3β inhibitor (BIO) for 24 h. Quantification of the normalized protein expression in the right panel. Statistical differences were determined by two-tailed Student t test. N = 3 independent experiments. All data are presented as mean ± SEM. *p < 0.05; **p < 0.01; **p < 0.001