Nvp tae684

TAE684 (NVP-TAE684) (HY-10192, MedChem Express), a specific inhibitor of Ltk and Alk (Colanesi et al., 2012 (link); Galkin et al., 2007 (link); Fadeev et al., 2016 (link); Rodrigues et al., 2012 (link)), was diluted in dimethylsulphoxyde (DMSO; Sigma-Aldrich Louis, MI, USA) to a final concentration of 6 mM. Clownfish juveniles were treated in 0,005% DMSO with 0.6 µM TAE684 or without (controls). For each condition, 3 juveniles were treated in 2 L fish medium in a beaker. 400 ml solution was changed every day.

Crizotinib, NVP-TAE684 and LDK-378 for use in tissue culture studies were purchased from Selleck Chemicals (Houston, TX), dissolved in DMSO and stored at −20C. For in vivo use, Crizotinib was obtained from Aok Bio (Shanghai, China), and NVP-TAE684 and LDK-378 were sourced from MedChemExpress (Monmouth Junction, NJ). The purity of all drugs were greater than 95%, as assessed by LC-MS analysis. ALK inhibitors were suspended in capryol 90 (Gattefosse Corp., Paramus, NJ) for oral gavage of animals. Vincristine (V), actinomycin D (A) and cyclophosphamide (C) were all obtained from the St. Jude pharmacy and were dissolved in sterile saline. They were administered by i.p. injection using a schedule deemed efficacious in pediatric patients (V given weekly at 0.38mg/kg; A given once at 0.5mg/kg; C given once at 125mg/kg).
Antibodies for ALK (31F12 mouse monoclonal, cat# 3791) and G3PDH (D16H11 rabbit HRP-conjugated monoclonal, cat# 8884) were purchased from Cell Signaling (Danvers, MA). siRNA targeting ALK and GFP-tagged RNA used as a control for transfection efficiency were obtained from OriGene (Rockville, MD). The former consists of 3 unique 27mer siRNA duplexes targeting different regions of the ALK mRNA and scrambled siRNA were used as negative controls.