Fig. 5.

HGF protease inhibitors reduce HGF maturation and resensitize CC-HGF cells to cetuximab. A Two hundred thousand CC and CC-HGF cells were grown on plastic and four days later were incubated with inhibitors indicated (50 μM) for an additional 48 h. Conditioned media were then subjected to immunoprecipitation with GFP-Trap Magnetic beads and resolved on SDS-PAGE and probed with HGF antibody recognizing β-chain as indicated. Expected full-length (inactive) and cleaved GFP-bound βfragment (indicating protease activity) HGF isoforms are depicted as schematic on left of observed bands. Corresponding cellular lysates were immunoblotted for indicated proteins as depicted in lower panels. B Two thousand CC-HGF cells were seeded in type I collagen and incubated with cetuximab (CTX, 3 μg/ml) or VD2173 (25 μM) for 14 days. Colony counts are plotted as mean ± SEM; * indicates statistically significant differences (one-way ANOVA with Tukey's HSD post hoc test, p < 0.05). C One hundred thousand CC-HGF cells were cultured in type I collagen for seven days and incubated with CTX (3 μg/ml) and/or VD2173 (25 μM) for 48 h and then lysed and resolved on SDS-PAGE followed by immunoblotting for proteins as indicated