07 min, MS : m z 195. 1 The 3 benzoic acid methyl ester was suspended in toluen

IGF 1 was purchased from Peprotech, LY294002 and PP242 were purchased from Sigma, Stock solutions Ivacaftor 873054-44-5 of these agents were subsequently diluted with serum free RPMI 1640 medium prior to use. In all experiments, the final concentration of DMSO did not exceed 0. 1%. MTT colorimetric survival assay Cell viability was monitored by 3 2,5 diphenyltetrazolium bromide assay. Briefly, cell lines and primary leukemic cells were seeded in 96 well plates and treated with SNS 032 for the indicated times. The end of culture period, 20 ul of MTT solution was added to each well and then the samples were incubated at 37 C for 4 h. The absorbance of the reaction was measured at 570 nm by spectrophotometry. IC50 values were calculated.<br><br> Panobinostat LBH589 Colony forming assay The effects of SNS 032, perifosine, or combination on the leukemia colony formation in methylcellulose medium were examined using leukemic colony assay as previously described, Briefly, leukemic cells in 600 uL of methylcellulose solution were incubated in the presence of the agents or an equivalent amount of medium at 37 C in a humidified atmosphere with 5% CO2. Primary leukemic cells were cultured in methylcellulose medium containing recombinant human stem cell factor, granulocyte macrophage colony stimulating factor, and interleukin 3 at 2 × 104 cells dish. After 7 days, CFU Ls that contain 40 cells were scored manually under a light microscope, For colony assay of human normal bone marrow cells, 3 U mL rh erythropoietin, 50 ng mL rhSCF, 30 ng mL rhGM CSF, and 10 ng mL rhIL 3 were added to the methylcellulose medium.<br><br> The colonies were counted under a microscope on day 12 of culture. Flow cytometric analysis HL 60, KG 1 and HEL cells were treated with SNS 032 at concentrations between 50 and 200 nM for 24 h. Apoptotic cells were quantified by Annexin V FITC and propidium LY2109761 価格 iodide double staining using a detection kit purchased from Biouniquer according to the manufacturers instructions. Western blot analyses Cells were incubated for 6 h in the presence or absence of the drugs. The cells were then lysed at 4 C in lysis buffer. Protein concentration was determined by the bicinchoninic acid method. The total protein was used for Western blot analysis as previous described, Aliquots containing 50 ug proteins were separated on sodium dodecyl sulfate polyacrylamide gels containing 6 12% acrylamide gradients and then transferred to polyvinylidene difluoride membranes, The membranes were blocked for 2 h in Tris buffered saline containing 0.<br><br> 1% Tween and 5% nonfat dry milk and then incubated with primary antibodies overnight at 4 C, followed by incuba tion with secondary antibodies conjugatesd with fluores cent dyes for 2 h at room temperature. After washing three times, the membranes were incubated with anti rabbit mouse IgG conjugated to horseradish peroxidase. The results were visualized with the ECL detecting kit. All primary antibodies were purchased from Cell Signal ing Technology, except the human anti RNA poly II, RNA poly II CTD phospho Ser2 and phospho Ser5, and phospho Akt, PI3K p110 primary antibodies.