Fig. 3

Receptor-antibody hybrid proteins inhibit HGF-dependent MET activation and biological responses. A - Hybrid proteins inhibit HGF-induced MET phosphorylation. A549 cells were incubated with 1 μM of each fusion protein for 24 h and then stimulated with 100 ng/ml of HGF for 15 min. p145 pMET, phosphorylated MET β-chain; p145 MET, MET β-chain; p90, HSP90. B - Hybrid proteins inhibit HGF-induced cell scattering. Representative images of HPAF-II cells treated with 0.2 or 2 μM of each fusion protein for 24 h and then stimulated with 6.25 ng/ml of HGF. NT, not treated cells. Magnification, 40x C - Cell scattering monitored in real time using an X-CELLigence RTCA device and expressed as normalized Cell Index (CI). HPAF-II pancreatic cancer cells were plated in E-plate wells, and then treated with 6.25 ng/ml HGF alone or in presence of 3 μM of the hybrid proteins. D - Hybrid proteins inhibit HGF-induced cell invasion. HPAF-II cells were stimulated with 12.5 ng/ml of HGF and treated with 1 μM of each hybrid protein. Graph represents the percentage of invasion in comparison with the HGF-stimulated cells. Each point is the mean of values in duplicate ± SD. One representative image/group of the cells migrated through the matrigel layer is showed below the graph. NT, not treated cells. Magnification, 25x. E - Hybrid proteins impair the viability of patient-derived colon cancer spheroids. M049 colon spheroids were pre-incubated with 20 ng/ml HGF and treated with increasing concentrations (0–700 nM) of each hybrid. Cell viability was determined by measuring cellular ATP. Graph represents the percentage of viability in comparison with the HGF-stimulated control. Each point is the mean of values in triplicate ± SD. ***, p≤ 0.001; **, p≤ 0.01; *, p≤ 0.05, ns: not significant. Data reported in the figure are representative of one experiment out of three