R428 bgb324

Axl tyrosine kinase inhibitors R428 (BGB324) and SGI-7079 were purchased from Selleck Inc. R428 and SGI-7079 specificities for Axl have been previously evaluated [21 (link), 24 (link)]. They were dissolved in DMSO at the concentration of 1 mM for in vitro studies while formulated in 0.5% hydroxypropylmethylcellulose plus 0.1% Tween 80 for in vivo studies. Transfection agent Lipofectamine 2000 was obtained from Invitrogen. Scramble control Axl-targeting siRNA was synthesized by Shanghai GenePharma Inc with the following sequences: siAxl-1: 5′-GCCAGATGAAATCCTCTAT-3′, siAxl-2: 5′-CGAGGTACTTATGGATATA-3′; siNC: 5′-AATTGTACTACACAAAAGTAC-3′. Anti-mouse PD-1 (RMP1–14), CD4 (GK1.5), CD8 (2.43), NK 1.1 (PK136) and their isotype control (2A3) antibodies were purchased from BioXCell. Primary rabbit anti-mouse/human Axl antibody (ab32828) and secondary FITC-labelled goat anti-rabbit antibody (ab6717) were obtained from Abcam. PE-conjugated anti-mouse Axl (FAB8541P), Mer (FAB5912P) and rat IgG2a isotype control (IC006P) antibodies were purchased from R&D Systems. The fluorescence labeled monoclonal antibodies (mAbs) for FACS analysis were all from BD Bioscience.

AM38, DBTRG-05MG, LN229, NMC-G1 and U87MG cells were purchased from ATCC. 8MGBA, 42MGBA were obtained from DSMZ. ATRT lines BT12, BT16 and 794 was kindly provided by Dr N. Foreman (University of Colorado). CHLA-07-BSGBM was the provided by the courtesy of Dr Anat Erdreich-Epsten (Children's Hospital Los Angeles). SF188, SF9744, SF9841 and SF9867 were obtained from the Brain Tumor Research Center (University of California, San Francisco).
Wild-type and K567RAxl in pcDNA3.1 (+) vector was kindly provided by Dr Trever Bivona (Helen Diller Cancer Center, UCSF) [30 (link)]. pcDNA3.1-v5/His plasmids containing human full-length EGFR has been described previously [32 (link)]. PLX4720 was provided by Plexxikon Inc (Berkeley, CA, USA) and HKI-272 (Neratinib) was purchased from TSZ Scientific LLC (MA, USA). Axl inhibitors foretinib (GSK1363089) and R428 (BGB324) were purchased from Selleckchem (Houston, Texas, USA) and Axon Medchem (Reston, Virginia, USA) respectively. All drugs were dissolved in dimethylsulfoxide (DMSO) at 10 mM and stored at −20°C. The final DMSO concentration in all experiment was less than 0.1% in medium.

MDAMB231 cells were purchased from ATCC and grown in DMEM (Gibco) media supplemented with 10% FBS, 1% Pen/strep, and 1% Glutamax Supplement (Thermo Fisher) and maintained at 37 °C in 5% CO₂. SUM159 cells were purchased from Asterand Bioscience and grown according to manufacturer’s suggestion. Recombinant human EGF was used at 10 nM. Batimastat (BB94, Tocris Bioscience) was used at 10 μM. Selumetinib (AZD6244, Selleck Chem) was used at 1.5 μM. Binimetinib (Mek162, ARRY-162, Selleck Chem) was used at 1.2 μM. PD0325901 (Selleck Chem) was used at 33 nM. Ulixertinib (BVD-523, VRT752271, Selleck Chem) was used at 30 nM. DEL-22379 (Selleck Chem) was used at 5 μM. GDC-0994 (Selleck Chem) was used at 30 nM. JQ1 (Tocris Bioscience) was used at 0.2 μM. R428 (BGB324, Selleck Chem) was used at 1 μM. DMSO at matched concentrations was used as all controls.