MTOR; Raptor: Regulatory associated protein of mTOR; TK: Tyrosine kinase; TKI: Tyrosine kinase inhibitor; TKD: Tyrosine kinase domain; XTT: 2,3Bis(2methoxy4nitro5sulfophenyl)SMCC web 2Htetrazolium5carboxanilidsodium salt. Competing interests Dr. KampaSchittenhelm: no conflicts. Dr. Heinrich Consultant Novartis, MolecularMD, Research funding: Novartis, Ariad, Imclone, AROG, Equity interest: MolecularMD. Figen Akmut: no conflicts. Katharina Henriette Rasp: no conflicts.Cells had been treated in dilution series with the respective modest molecule inhibitor. Translocation of phosphatidylserine from the inner towards the outer leaflet on the plasma membrane as an early indicator of apoptosis was analyzed employing an Annexin Vbased assay (Immunotech, Marseilles, France) and also a FACScaliburflow cytometer loaded with CellQuestanalysis Software (BD, Heidelberg, Germany) [35]. Cellular proliferation was measured making use of an 2,3bis [2methoxy4nitro5sulfophenyl]2Htetrazolium5carboxanilide inner salt (XTT) ased assay (Sigma) as described previously [35].Cell cycle assayA propidium iodidebased flow cytometry assay was assessed as described previously [56]. In brief, a propidium iodide stain assay is applied to segregate cells according to the DNA content, that is graphically shown inside a histogram plot (high content in G2M, intermediate content material in Sphase, low content material in G1G0 and lowest content material in deadapoptotic cells, which defines a subG1G0 fraction),Data analysisLinear regression dose effect plots to calculate IC50s were computed with values in in between upper and reduce threshold doses of minimalmaximal dose effects using Calcusyn Software (Biosoft, Cambridge, UK), which is depending on equations provided by Chou and Talaly [37].KampaSchittenhelm et al. Molecular Cancer 2013, 12:46 http:www.molecularcancer.comcontent121Page 17 ofBarbara Illing: no conflicts. Dr. Hartmut D ner: Consultant: Novartis, Celgene, Boehringer Ingelheim, Ambit. Dr. Konstanze D ner: Consultant: Novartis. Dr. Schittenhelm: no conflicts. Authors’ contributions KS designed study, Development Inhibitors Reagents performed research, analyzed data and wrote the paper. MH analyzed data, and wrote the paper. FA performed analysis and analyzed data. KR performed investigation analyzed information. BI performed study analyzed data. HD analyzed information, and wrote the paper. KD analyzed data, and wrote the paper. MS designed study, performed analysis, analyzed data and wrote the paper. All authors read and approved the final manuscript. Acknowledgements We thank the core facilities of the Medizinische Universit sklinik T ingen for exceptional technical help. After 24hours of incubation at 37 , the cells werefixedwith4 paraformaldehyde,stainedwith0.1 crystal violet, and counted below a microscope at 100magnification.two.9Cell proliferation assayAll the cells had been seeded in 96well plates at a density of 1 103 properly and counted each day for the following 5 days. At the same time each and every day, the cells had been incubated with ten LsterileMTTdye(5mg mL) at 37 for 4hours. Soon after aspiration with the medium, the cells werelysedwithDMSO.Theabsorbanceat490nmineachwellwas recorded applying a microplate reader.two.12Flow cytometry analysis in the cell cycleThe cells had been cultured in serumfree medium for 24 hours then culturedinmediumwith10 FBSfor24hours.TheindicatedcancerSHI et al.cellswerecollectedandfixedwithprecooling75 ethanolat4 . Thefollowingday,the75 ethanolwasremoved,andthecellswere incubated in propidium iodide option (100 gmL) at space temper ature for 20 minutes in the dark.