Crizotinib

Human LAC cell lines A549 (ATCC CCL-185), H1975 (ATCC CRL-5908), HCC827 (ATCC CRL-2868), and H358 (ATCC CRL-5807) were cultured in RPMI medium containing 10% fetal bovine serum as previously described [34 (link)]. Growth factors HGF (R & D Systems), and anti-cancer drugs Gefitinib (Cell Signaling), Crizotinib (Cell Signaling), and Cyclopamine (Santa Cruz) were added to the culture medium in conditions as indicated in Figure legends.

Gefitinib (#G-4408), erlotinib (#E-4007), trametinib (#T-8123), crizotinib (#C-7900) and paclitaxel (#P-9600) were purchased from LC Laboratories (Woburn, MA, USA). Cisplatin (#479306) was purchased from Sigma Aldrich (St. Louis, MO, USA). 2D and 3D cultures were incubated in the presence of the drugs for 72 hours. Cell viability was determined using the commercially available alamarBlue^® assay kit (Thermo Fisher Scientific, Madison, WI, USA). Apoptosis was analyzed using the Caspase-Glo^® 3/7 assay multiplexed with the MultiTox-Fluor GF-AFC life stain (Promega, Madison, WI, USA). Luminescence and fluorescence intensities were measured using the Paradigm detection platform (Molecular Devices, Sunnyvale, CA, USA). Statistical analysis was done using the GraphPad Prism Software 7.03 (GraphPad Software Inc., La Jolla, CA, USA). For the in situ analyses of apoptosis in cocultures the cells were incubated in the presence of 50 nM Gefitinib or 20 nM crizotinib for 24 hours and processed for immunofluorescent microscopy using the cleaved caspase-3 antibody (Cell Signaling Technology, #9661) that specifically detects apoptotic cells.