Celltiter glo atp based assay

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.