Vs 4718

VS-4718 (#S7653), VS-6063 (#S7654), PF-562,271 (#S2890), GSK2256098 (#S8523), vorinostat (#S1047), panobinostat (#S1030), staurosporine (#S1421), and paclitaxel (#S1150) were purchased from Selleck Chemicals. Chemical structures are shown for the FAK kinase inhibitors (VS-4718, VS-6063, PF-562,271, and GSK2256098) and HDAC inhibitors (vorinostat and panobinostat) in Supplementary Fig. 1A. Compound libraries were from BioAscent. Antibodies used were purchased from Cell Signaling Technology (FAK phosphorylated tyrosine-(pY)397, #3283; FAK, #3285; GAPDH, #2118; YAP, #14074; YAP phosphorylated serine-(pS)127, #13008; histone H3, #9715; histone H3 acetylated lysine-56 (K-Ac), #4243; Cdc37, #4793; Cdc37 pS13, #13248; Src, #2109; Axl #8661) or Abcam (YAP, #EP1674Y).

Human NSCLC cell lines and human bronchoalveolar cells were provided by Dr. John Minna (UT Southwestern Medical Center) and cultured as described (27 (link), 28 (link)). A549 and H460 cells expressing vector control or LKB1 were previously described (17 (link)). All cells were mycoplasma free and were identified by DNA fingerprinting. Supplementary Table 1 shows the mutations that they harbor according to COSMIC: Catalogue of Somatic Mutations in Cancer (Cosmic; cancer.sanger.ac.uk). FAK inhibitors PF-562,271 and VS-4718 were obtained from Selleckchem and Verastem, Inc., respectively (29 (link), 30 (link)). Inhibitors were added to mid-log phase cell cultures at the indicated concentrations. All other chemicals were purchased from Sigma-Aldrich.

LNCaP, MDAPCa2b, 22Rv1, and PC3 cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). Cells were tested for mycoplasma contamination. LNCaP, 22Rv1, and PC3 were cultured in RPMI-1640 (R8758, Sigma-Aldrich, St. Louis, MO, USA), 10% fetal bovine serum (FBS) (F2442, Sigma-Aldrich), 5% CO2, at 37°C, 1% penicillin/streptomycin (#15140122, ThermoFisher Scientific, Waltham, MA, USA). For castrated condition, cells were cultured in RPMI-1640 without phenol-red (#11835030, ThermoFisher Scientific), 5% CSS (#F6765, Sigma-Aldrich). MDAPCa2b were maintained in F-12K (#21127022, ThermoFisher Scientific) Supp.mented with 20% FBS, 25 ng/mL cholera toxin (#C8052, Sigma-Aldrich), 10 ng/mL mouse EGF (#354010, Corning, Corning, NY, USA), 5 μM phosphoethanolamine (#P0503, Sigma-Aldrich), 100 pg/mL hydrocortisone (#H0135, Sigma-Aldrich), 45 nM sodium selenite (#9133, Sigma-Aldrich), and 5 μg/mL human recombinant insulin (#12585–014, Life Technologies, Carlsbad, CA, USA). For castrated conditions, 20% of CSS was used instead of FBS. Carbachol was purchased from Sigma Aldrich (#C4382). Enzalutamide (#S1250) and VS-4718 (S7653) were purchased from Selleckchem (Houston, TX, USA), Clozapine N-oxide (CNO) was purchased from Sigma-Aldrich (#C0832), thrombin was purchased from Millipore Sigma (605190), LPA was purchased from Sigma-Aldrich (L7260).

To determine the effects of perturbations on Ewing sarcoma cell viability, cells were plated in 384-well plates at a concentration of 1000 cells per well in 50 μL of medium. Cell viability was measured by adding 10 μL of Cell-Titer Glo ATP-based assay (Promega). Luminescence was read using the FLUOstar Omega microplate reader (BMG LabTech). VS-4718, GSK-1070916, and NVP-AEW541 were obtained from Selleck for in vitro treatment of cells. Cells undergoing apoptosis were stained with Annexin V using the Apoptosis Detection Kit-APC (eBioscience) and cellular DNA content was measured by propidium iodide staining (Invitrogen). For intracellular phospho-protein staining, cells were fixed and permeabilized using the BD Cytofix/Cytoperm Kit (BD Biosciences) and stained with phycoerythrin (PE) anti-phospho-S6 (S240, BD Biosciences) and analyzed by flow cytometry. A minimum of 10,000 stained cells were analyzed in all flow cytometry experiments. All experiments testing viability, apoptosis, cell cycle, and measurements of phosphorylation of S6 were performed with two or more experimental replicates and each experiment repeated a minimum of two times. Experiments shown are representative of experimental and biologic replicates.