Another important issue in designing clinical trials for MTAs is patient enrich

At 24 h after transfection, EOL 1 cells were adjusted to 2×105 ml Ivacaftor 臨床試験 and exposed to ponati nib treatment, then underwent cell death assay and immunoblotting. Luciferase assay EOL 1 cells were transfected with TOPflash or FOPflash plasmid and pEFRenilla luc by electroporation. At 24 h, cells were incubated with or without ponatinib for 24 h. Luciferase activity was then measured with the dual luciferase assay kits as described. GG 3. In brief, oligonucleotides for TCF LEF were la beled with biotin by use of the biotin 3 end DNA label ing kit. In total, 5 ug of nuclear extracts was incubated for 20 mins with 1 ug ul poly and biotin end labeled target nucleotides in 20 ul reaction mixtures. The resulting bound complex was separated from free oligonucleotides on 6% native polyacrylamide gel and transferred to a nylon membrane.

After cross linking, blocking, and reacting with substrates, the membranes were exposed to X ray film to detect biotin labeled DNA. オーダー LBH589 The binding specificity was examined by competition with a 200 fold excess of the unlabeled oligonucleotide probe. Transmission electron microscopy The cells were treated with or without ponatinib, and then fixed with 2% glutaraldehyde plus 2% paraformalde hyde in 0. 1 M cacodylate buffer. After washing, and postfixing, the samples were dehydrated and embed ded in Spurrs low viscosity medium. Ultrathin sec tions of the samples stained with uranyl acetate and lead citrate were examined under a JEM 1010 transmission electron microscope. Immunofluorescence staining EOL 1 cells were treated with or without ponatinib for 24 h, and then harvested by use of Cytospin onto glass slides.

Immunofluorescence staining was as described. DyLight 488 conjugated goat anti mouse immuno globulin was purchased from Pierce Biotechnology. Apoptosis assessment Apoptosis LY2109761 msds was evaluated by using an Annexin V fluorescein isothiocyanate or Annexin V phycoerythrin apoptosis detection kit and analyzed by using a FACSCalibur flow cytometer. Cell cycle analysis Control or ponatinib treated cells were fixed with 66% ethanol overnight. DNA content was analyzed by flow cytometry after cells were stained with 50 ug ml PI and 2. 5 ug ml RNase in PBS solution for 30 mins. Tumor xenograft experiments Male nu nu BALB c mice were bred at the animal facil ity of Sun Yat sen University.

An amount of 1×107 BaF3 T674I PDGFR cells supplemented with 50% matri gel was inoculated subcutaneously on the flanks of 4 6 week old male nude mice. Tumors were measured every other day with calipers. Tumor volumes were calculated by the following formula, a2 × b × 0. 4, where a is the smallest diameter and b is the diameter perpendicular to a. Ponatinib was initially dissolved in DMSO and then adjusted to the appropriate doses with vehicle, and imatinib was dissolved in sterile double distilled water. Mice in each group were treated once daily by oral gavage with ponatinib, imatinib or the same amount of vehicle. The body weight, feeding behavior and motor activity of each animal were monitored as indicators of general health. Tumor xenografts were immediately removed, weighed, stored and fixed after animals were killed.