Tae684

The inhibitors of ALK tyrosine kinase, crizotinib was obtained from LC Laboratories (Woburn, MA, USA), and ceritinib and TAE684 from Selleck Chemicals (Houston, TX, USA). The Mps1 inhibitor AZ3146 was obtained from Adooq Bioscience (Irvine, CA, USA), and the reversible CDK1 inhibitor RO-3306 from Selleck Chemicals. These inhibitors were dissolved in DMSO (Nacalai Tesque, Kyoto, Japan).

The H2228 cell line was obtained from the American Type Culture Collection (Rockville, MD), and the H3122 cell line was a gift from Adi F. Gazdar (UT Southwestern, Dallas, TX). Cells were cultured in 10% fetal bovine serum (FBS), 100-U/mL penicillin, and 100-mg/mL streptomycin (Invitrogen, Carlsbad, CA) at 37°C in an atmosphere with 5% CO₂. Crizotinib, TAE684, ceritinib, alectinib, gefitinib, afatinib, PHA 665752, and AUY922 were purchased from Selleck Chemicals (Houston, TX). EGF and IGF-1 were purchased from Calbiochem and Sigma–Aldrich (St. Louis, MO), respectively. BI 836845 was kindly provided by Boehringer Ingelheim (Vienna, Austria).

AT9283, AZD4547, AZD6244, BGJ398, crizotinib, pictilisib, ibrutinib, PD173074, ruxolitinib, TAE684, and gandotinib were all purchased from Selleck Chemicals, AUY922, dovitinib, everolimus, gefitinib, mubritinib, saracatinib, sunitinib, and ZSTK474 were from LC Laboratories, CH5424802 was from Active Biochem, SB525334 was from Tocris, and fedratinib was from Axon Medchem. The human gastric cancer cell line SNU-16 was obtained from ATCC and was cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum according to the manufacturer’s instructions.

Cells were seeded in 24 well plates and grown for 2 days in full medium followed by 24h in FCS-free medium before treatment with the same concentrations of ligands and inhibitors.
All inhibitors were dissolved in DMSO and cells were treated for 90 minutes at the following concentrations: GDC0941 (1 μM, Selleck Chemicals), AZD6244/Selumetinib (10 μM, Selleck Chemicals), MK2206 2HCl (10 μM, Selleck Chemicals), Rapamycin (10 μM, Selleck Chemicals), Sorafenib (10 μM, Selleck Chemicals), GS-4997 (10 μM, Selleck Chemicals) and TAE684 (10 μM, Selleck Chemicals).
The cells were treated for 30 minutes (60 minutes after inhibitor treatment) with ligands in a 0,1% PBS/BSA carrier solution at the following concentrations: EGF (25 ng/mL, Peprotech), PDGF (10 ng/mL, Peprotech), NGF (50 ng/mL, Peprotech) and IGF1 (100 ng/mL, Peprotech).
The cells were then lysed using BioRad Bio-Plex Cell Lysis Kit and measured using the Bio-Plex MAGPIX Multiplex Reader with a custom kit from ProtAtOnce with analytes p-cJUN (S63), p-p38 (T180/Y182), p-AKT (S473), p-ERK1/2 (T202/Y204,T185/Y187), p-MEK1 (S217/S221), p-S6K (T389) and p-RSK1 (S380). The p-RSK1 (S380) readout was discarded because of a low dynamic range.
The same procedure and analytes were used for the other perturbation assays in this paper. Refer to the main text for the exact inhibitors and concentrations used for each experiment.

Human embryo kidney (HEK293) cells do not express STAT6 and the human lung adenocarcinoma cell line H2228, expressing STAT6 and harboring the EML4-ALK variant 3 fusion gene, were obtained from the American Type Culture Collection. The NIH3T3 mouse embryonic fibroblasts were obtained from the Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in Gibco™ Dulbecco’s modified Eagle medium supplemented with 10% FBS and 1% penicillin–streptomycin at 37 °C with 5% CO₂. IL4 and IL6 were purchased from PeproTech (USA), diluted in sterile ultrapure water as indicated by the manufacturer, and frozen in aliquots at − 80 °C. The small molecule inhibitors TAE684 and SH-4-54 were purchased from Selleck Chemicals (USA), diluted, and stored according to the manufacturer’s instructions.