Amg337

The primary antibodies used in this study were Myc-Tag (9B11), CDCP1, Met (D1C2), phospho-Met (Tyr1234/1235) (D26), phospho-tyrosine (P-Tyr-1000), phospho-Src (Tyr416), phospho-Akt (Ser473), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), Vav2 (C64H2), and Rab7 (D95F2) obtained from Cell Signaling Technology. Anti-GAPDH antibody was purchased from Santa Cruz Biotechnology. Anti-EEA1 and Anti-Rac1 antibodies were purchased from BD Biosciences. Anti-M6PR antibodies were purchased from Thermo Fisher Scientific. Anti-ARHGEF7 antibody was prepared by immunizing rabbits with C-terminal fragments. Dynamin inhibitor Dynasore, PI3K inhibitor LY294002, and SFK inhibitor Dasatinib were purchased from Abcam. ROCK inhibitor Y27632 was purchased from Cayman chemical. RAC1 inhibitor was purchased from Merk. MET inhibitors, AMG337 and Crizotinib, were purchased from Selleck Chemicals.

Growth Factor rescue experiments were performed in DiFi and LIM1215 colorectal cancer cell lines treated with CET (provided by Merck KG), AMG-337 and BGJ-398 (Selleckchem), FGF1, FGF2, TGFβ1, TGFβ2 and TGFβ3 (RnD Systems) and HGF and FGF10 (Peprotech) for 5 days (7 days for FGF10). Treatments were replenished with fresh media after 3 days in 7 day assays. EGFR mutant experiments were performed in LIM1215 cells. Cells were treated with CET for 5 days. DiFi and LIM1215 cells were seeded in standard media or CAF CM and treated with CET for 5 days. All experiments were performed in 6 replicates. Viability was assessed using CellTiter Blue reagent (Promega) for all assays.