Foretinib

The cells were incubated with diverse concentrations of EGFR inhibitor lapatinib (Santa Cruz Biotechnology, CAS 231277–92‐2), MET inhibitor crizotinib (Santa Cruz Biotechnology, sc‐3,564,471) or foretinib (Santa Cruz Biotechnology, CAS 849217–64‐7) separately or in combinations (crizotinib/lapatinib and foretinib/lapatinib) for 24 or 48 h. The inhibitors' concentrations used in the experiments (2 or 5 μM crizotinib, 2 or 5 μM foretinib and 5 or 7.5 μM lapatinib) were selected based on previous XTT experiments (data not shown). For each assay, cells were stimulated with 5 nM EGF (Corning) and 30 ng/mL HGF (Sigma Aldrich) to imitate conditions present in the melanoma microenvironment. Cells incubated only with growth factors and 0.1% DMSO (inhibitors solvent) were treated as a control.

Rabbit polyclonal anti-cortactin antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Alexa Fluor 568-conjugated phalloidin, secondary anti-rabbit antibodies conjugated with Alexa Fluor 488, gelatin conjugated with FITC, fetal bovine serum (FBS), trypsin, glutamine, penicillin/streptomycin, and DMEM were purchased from Invitrogen (Carlsbad, CA, USA). Dako fluorescent mounting medium was obtained from Dako (Agilent, Santa Clara, CA, USA). The epidermal growth factor (EGF) and rat-tail collagen (acid-extracted, non-pepsin treated) type I were obtained from BD Biosciences (Agilent, Santa Clara, CA, USA). Cell Proliferation Kit II was purchased from Roche (Basel, Switzerland). Antibodies directed against EGFR (SC-03), MET (SC-10), AKT1/2/3 (SC-8312), and phospho-AKT1/2/3 (S473; SC-135651) were purchased from Santa Cruz Biotechnologies. Anti-phospho-EGFR (Y1069; catalogue no. 3777), anti-phospho-MET (Y1234/Y1235; no. 3077), anti-ERK1/2 (no. 9102), and anti-phospho-ERK1/2 (T202/Y204; no. 9101) antibodies were from Cell Signaling Technologies (Danvers, MA, USA). HGF was obtained from Sigma Aldrich (Saint Louis, MO, USA), foretinib from Santa Cruz Biotechnologies, and lapatinib from Selleckchem (Houston, TX, USA). All other chemicals were classified as analytical grade reagents.

Rabbit polyclonal anti-cortactin, mouse anti-phosphorylated Src, and mouse anti-GAPDH protein (glyceraldehyde 3-phosphate dehydrogenase) antibodies were purchased from Santa Cruz Biotechnology. Mouse anti-Src antibodies were obtained from Merck Milipore. Alexa Fluor 568–conjugated phalloidin, secondary anti-rabbit antibodies conjugated with Alexa Fluor 488, gelatin conjugated with fluorescein (FITC), fetal bovine serum (FBS), trypsin, glutamine, and penicillin/streptomycin/amphotericin B solution were obtained from Invitrogen, while DMEM from IITD PAN, Wroclaw. Dako Fluorescent Mounting Medium was purchased from Dako. EGF and Matrigel were obtained from BD Biosciences, while HGF from Sigma. Foretinib was purchased from Santa Cruz Biotechnologies; lapatinib and gefitinib from Selleckchem. Goat anti-mouse antibodies conjugated with horseradish peroxidase were obtained from Cell Signaling Technologies. All other chemicals were classified as analytical grade reagents.