Ag 1024

Cortical neurons were treated with inhibitors or ligands 4 h after plating and were fixed 33–38 h after treatment for analysis. For IGF‐1R, inhibitors or activators were added in starvation medium (serum‐free Neurobasal medium, supplemented with 2% B27 minus insulin, 1% L‐glutamine, 50 IU/ml penicillin, and 50 μg/ml streptomycin). The following inhibitors and ligands were used: TAE684 (NVP‐TAE684, Selleckchem, #1108), crizotinib (PR‐02341066, Selleckchem #1068), LY 294002 (Tocris), BKM120 (Buparlisib) (Selleckchem, #S2247), AG1024 (Selleckchem #S1234), PPP (CAS 477‐47‐4, Santa Cruz, scbt #24008), and recombinant murine IGF‐1 (Peprotech).
ALKAL2‐containing conditioned medium (ACM) was generated as described previously (Guan et al, 2015 (link)) with slight modifications. Briefly, HEK293T cells, in 10 cm plates, were transfected with 8.0 μg pcDNA3‐FAM150B‐HA using the calcium phosphate method. After 16 h, media was replaced with starvation medium (DMEM, supplemented with 0.2% FBS, 50 IU/ml penicillin, and 50 μg/ml streptomycin) and ALKAL2‐conditioned medium was collected after 36 h. Cortical neurons were treated with CCM and ACM, supplemented with 2% B27 and 1% N2. Activation of wild‐type endogenous ALK was confirmed in IMR‐32 human neuroblastoma cells.

EGFR-TKI-sensitive PC-9 cells were gifted by Prof. J. Xu and Dr. M. Liu from Guangzhou Medical University (China), and HCC827 cells were provided by the American Type Culture Collection. EGFR-TKI acquired resistant PC-9GR cells were gifted by Prof. J. Xu and Dr. M. Liu from Guangzhou Medical University (China), and H1650-M3 cells were kindly provided by Dr. R. Sordella from Cold Spring Harbor Laboratory. EGFR-TKI primary resistant H1975 cells were provided by the American Type Culture Collection. All the cells were cultured in the Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone; HyClone, Logan, UT, USA) with Earle’s salts and supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA), 2 mM L-glutamine solution (Thermo Fisher Scientific), 100 U/mL penicillin (HyClone) and 100 μg/mL streptomycin sulfate (HyClone) at 37°C with 5% CO₂ in the air and 90% humidity. Gef (Iressa), purchased from Tocris Bioscience (Ellisville, MO, USA), and AG-1024, purchased from Selleck Chemicals (Houston, TX, USA), were prepared in dimethyl sulfoxide (DMSO) to obtain a stock solution of 10 mmol/L. Met (Sigma-Aldrich Co., St Louis, MO, USA) was dissolved in deionized water, and a 1 M stock solution was prepared and stored in aliquots at −20°C. The use of gifted cell lines was approved by ethics committee of the Third Military Medical University.