Cell lines used, with their respective mutations, are listed in Supplementary Table S1 . The murine interleukin 3 (Il-3)-dependent pro-B-Cell line, Ba/F3, was used as a model to obtain ALK-dependent cells, owing to its potent growth capacity and use of kinase oncogene signalling53 (link). Cells were grown in RPMI 1640 (Invitrogen) supplemented with 10% FCS, 100IU/ml penicillin/streptomycin, 2 mM L-glutamine and 25 mM HEPES (Life Technologies). Ba/F3 cells were cultured in the presence of 1 ng/mL Il-3 (213-13, Peprotech). Cells were cultured at 37 °C in a 5% CO2/95% O2 humidified environment. Mycoplasma testing (Lonza) and short tandem repeat genotyping were regularly performed.
Cells were treated for indicated time periods with inhibitory compounds: the ALK inhibitors, NVP-TAE-684 (0.32 µM, S1108, Novartis/SelleckChem), crizotinib (0.5 µM, PF-02341066, S1068, Pfizer/SelleckChem) or LDK-378 (0.2 µM, S7083, SelleckChem); the MEK inhibitors, U-0126 (8 µM, Sigma-Aldrich) or trametinib (0.05 µM, GSK1120212, S2673, SelleckChem); the dual PI3K/mTOR inhibitor, NVP-BEZ-235 (0.5 µM, S1009_3, SelleckChem) or the PI3K inhibitor pictilisib (0.5 µM, S1065, SelleckChem). Dimethyl sulfoxide (DMSO, VWR) was used to dissolve the compounds and served as vehicle control.
Cells were treated for indicated time periods with inhibitory compounds: the ALK inhibitors, NVP-TAE-684 (0.32 µM, S1108, Novartis/SelleckChem), crizotinib (0.5 µM, PF-02341066, S1068, Pfizer/SelleckChem) or LDK-378 (0.2 µM, S7083, SelleckChem); the MEK inhibitors, U-0126 (8 µM, Sigma-Aldrich) or trametinib (0.05 µM, GSK1120212, S2673, SelleckChem); the dual PI3K/mTOR inhibitor, NVP-BEZ-235 (0.5 µM, S1009_3, SelleckChem) or the PI3K inhibitor pictilisib (0.5 µM, S1065, SelleckChem). Dimethyl sulfoxide (DMSO, VWR) was used to dissolve the compounds and served as vehicle control.