Su11274

Antibodies that recognize c-Met, p-c-Met (Tyr1234/1235), ERK1/2, p-ERK1/2, AKT, p-AKT, E-cadherin, and vimentin were purchased from Cell Signaling Technology. Antibodies against PLXDC2 and ELK1 were ordered from Proteintech and Abcam, respectively. The c-Met inhibitor SU11274 and β-Actin antibody were obtained from Sigma-Aldrich. The secondary antibodies were purchased from Invitrogen. PE mouse anti-human CD133 and FITC mouse anti-human CD44 were obtained from BD Biosciences and used for flow cytometry. The c-Met inhibitor foretinib and crizotinib, AKT inhibitor AZD5363, and ERK1/2 inhibitor SCH772984 were obtained from SelleckChem. Cell viability was measured by alamarBlue Cell Viability Reagent (Thermo Fisher Scientific) following the indicated treatments. Western blotting, wound healing assays, cell proliferation, and colony formation assays were carried out as described previously (15, 16 (link)).

The following 43 reagents were used in this study: AMG 102 and panitumumab (Amgen); cercosporamide (BioAustralis); AKT inhibitor V (Calbiochem); AS-252424, bisindolylmaleimide, CGP 57380 and imatinib (Cayman Chemical); CTX ILK inhibitor (CRC); Avastin (bevacizumab) (Genentech/Roche); 4EGI-1, ABT-737, ABT-737 enantiomer, pazopanib, and retinoic acid (Santa Cruz Biotechnology); erlotinib, lapatinib, sorafenib, and temsirolimus (Scientifix); bortezomib and CYT387 (Selleck Chemicals); AKT-I-1/2, axitinib, bicalutamide, cilostazol, cyclopamine, DAPT, dasatinib, docetaxel, doxorubicin, floxuridine, fluorouracil, goserelin, ifosfamide, PD98059, ribavirin, salinomycin, SU11274, sunitinib, Tamoxifen, and vinblastine (Sigma); NSC 7908 (Tocris); and temozolomide (Wyeth).

Human lung cancer A549 and fibroblast HFL1 cells were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). A549 and HFL1 cells were maintained in PRMI 1640 and IMDM (Cellgro, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, USA) and antibiotics (penicillin, 100 U mL^-1; streptomycin 100 μg mL^-1) at 37°C in a humidified atmosphere of 5% CO₂, respectively. The cells were grown to approximately 80% confluence and collected for experiments. Furthermore, A549 cells (1000 cells/chamber) were cultured in maintenance medium alone, a mixture of maintenance medium and CAF in the presence or absence of human HGF neutralizing antibody (20 μg/ml; R&D systems, USA), or maintenance medium containing HGF (40 ng/ml, Sigma, USA) for 48h. Alternatively, the cells were cultured in a mixture of maintenance medium and CAF for 24 h and then cultured in the same medium or the medium containing an inhibitor for Met (SU11274, 5 μmol/L; Sigma, USA), PI3K/AKT1 (LY294002, 20 μmol/L; Beyotime, China) or GRP78 (BAPTA-AM-A1076, 20 μmol/L; Sigma, USA) for another 24 h.

HGF (recombinant human) was obtained from Millipore (Billerica, MA), EGF (recombinant human) from Sigma Chemical Co. (St. Louis, MO), IL-1α (recombinant human) from Biolegend (San Diego, CA), TGFβ (recombinant human) from R&D Systems Europe (Abingdon, UK), Human anti-HGF (MAB294) from R&D Systems Europe (Abingdon, UK), anti-alpha smooth muscle actin (αSMA) (BS66) from Nordic Biosite AB (Taby, Sweden) and SU11274 from Sigma-Aldrich (Oslo, Norway). Anti-phospho-EGFR (Tyr1173) was from Life Technologies/Invitrogen (Carlsbad, CA). Anti-phospho-Met (Tyr1234/1235), anti-phospho-Gab1 (Tyr627), anti-phospho-Akt (Ser473), anti-phospho-ERK1/2 (Thr202/Thr204), anti-vimentin (D21H3), anti-glial fibrillary acidic protein (GFAP) (GA5), anti-vinculin and anti-GAPDH were from Cell Signaling (Beverly, MA). Secondary goat anti-mouse and goat anti-rabbit IgG HRP-conjugated antibodies were purchased form Bio-Rad Laboratories (Hercules, CA). [³H]-thymidine (20–30 Ci/mmol) was from Perkin Elmer (Waltham, MA). All other chemicals used were of analytical quality.

The c‐MET inhibitor SU11274 (#S9820) and PARP inhibitor NU1025 (#N7287) were obtained from Sigma‐Aldrich. Both inhibitors were dissolved in DMSO and stored at − 80°C.

Y-27632 (Sigma), PP-3 and PP-2 (ABCAM) were used at 10 μM, Ly-294002 (Sigma) and PD-98059 (Enzo Life Sciences Inc.) at 20 μM, CGP-77675 (Sigma) at 5 μM, SU-11274 (Sigma) at 2 μM, Wortmannin (Sigma) at 0.1 μM, AG-1478 (Merck Millipore) at 200 nM, PF-562271 (Pfizer), PD-173074 (Tocris), EMD-121974 (Medkoo Biosciences), PF-573228 and PD-161570 (Sigma) at 1 μM.