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Caspase substrate solution containing the specific peptide substrate was then added to the supernatant and incubated for 2 h at 37 C before measurement by ELISA reader at 405 nm. RNA interference map キナーゼ 阻害剤 The siRNA againstNFB p65 was purchased from Santa Cruz Biotechnology, Santa Cruz, CA. For transfection with siRNAs, logarithmically growing cells were transfected with siRNA as instructed by the manufacturer. Western blot analysis HCT116 cells were incubated with TPL and ATF alone or in combination for 24 h, then lysed with RIPA buffer with protease inhibitor cocktail tablets, Supernatants were collected and protein con centration was determined by the Bio Rad protein assay method, Western blotting was performed according to standard protocols.<br><br> Proteins were separated by SDS PAGE and transferred onto nitrocellulose membranes that were blocked with 5% non fat milk in TBS containing 0. 1% Tween 20, and incubated with primary antibodies: p FAK, FAK, p JNK, c JUN, p c JUN, p AKT, uPAR, cleaved caspase Linifanib 分子量 3,NFB p65, BAX, BAD, BAK, cIAP, poly polymerase, tubulin, c FLIP L, GAPDH, Lamin B, Secondary antibodies were coupled to horseradish peroxidase, and were goat anti rabbit or goat anti mouse. Bound antibodies were then visualized with ECL plus Western blotting detec tion reagents, Signal intensity was quanti fied by densitometry using the software Image J, All experiments were done in triplicate and performed at least three times independently. Cell migration assay The effects of ATF, TPL or the combination on endothe lial cell and tumor cell migration were assessed by the transwell assay.<br><br> The cell migration assay was performed using transwell inserts as described previously, Before the experiment, HUVECs and HCT116 cells had been cultured in serum free medium with ATF, TPL or the combination for 16 h. Then the cells were harvested and resuspended in the same medium. 1 × 105 cells in a vole of 0. 1 mL were added to the upper chamber, and the lower chamber was filled with 0. LY3009104 dissolve solubility 6 mL of 20% FBS supplemented medium. After incubation at 37 C for 9 h, cells on the upper surface of the membrane were re moved. The migrant cells attached to the lower surface were fixed in 10% formalin at room temperature for 30 min, and stained for 20 min with a solution containing 1% crystal violet and 2% ethanol in 100 mM borate buffer, The number of cells migrating to the lower surface of the membrane was counted in five fields under a microscope with a magnification of × 100.<br><br> All groups of experiments were conducted in triplicate, and the cell number was counted by Image Pro Plus 6. 0 software. In vivo animal tumour model experiment Athymic nude mice were obtained from Shanghai Laboratory Animal Centre and housed under germfree conditions. Animal care and use were performed strictly in accordance with the ethical guidelines by Nanjing University Animal Care and Use Committee and the study protocol was ap proved by the local institution review board. HCT116 cells were injected subcutane ously into the dorsal flanks of mice. Tumour volume was monitored by measuring the two maximum perpen dicular tumour diameters with callipers every alternate day. All tumour bearing mice were divided randomly into 4 groups, and treatment was initiated on the 7th day when the volume of tumour reached a size of ap proximately 50 mm3.