In this study, we alter VEGF action by either over expressing, down regulating

They were dissolved in DMSO and stored at INNO-406 構造 −80 C. Soluble recombinant human TRAIL was purchased from Pepro Tech, Inc, The proteasome inhibitor MG132, the protein synthesis inhibitor cycloheximide and the GSK3 inhibitor SB216763 were purchased from Sigma Chemical Co, The neddyla tion inhibitor MLN4924 was provided by Millennium Pharmaceuticals, Inc. Expression plasmids in pCI vector carrying wild type and mutant human Mcl 1 were provided by Dr. X. Deng. Mouse monoclonal sur vivin and caspase 8 antibodies and rabbit polyclonal Bim, caspase 9 and PARP antibodies were purchased from Cell Signaling Technology. Mouse monoclonal caspase 3 antibody was purchased from Imgenex. Rabbit polyclonal Mcl 1, Bad, Bcl XL and SKP1 and mouse monoclonal Bcl 2, Cul 1 and tubulin antibodies were purchased from Santa Cruz Biotechnol ogy, Inc, GSK3 B antibody was pur chased from Upstate Millipore.<br><br> Mouse monoclonal Bax and rabbit polyclonal glyceraldehyde 3 phosphate dehydrogenase antibodies were purchased from Trevigen Inc, Both polyclonal and monoclonal actin antibodies were pur chased from Sigma Chemical Co. Cell lines and Lapatinib 溶解度 cell culture The human NSCLC cell lines used in this study includ ing those stably expressing ectopic Mcl 1 or survivin were described previously. A549 cells were re cently authenticated by Genetica DNA Laboratories, Inc. through analyzing short tandem repeat DNA profile; other cell lines have not been authenti cated. HCT116 wild type and HCT116 FBXW7 KO cell lines were kindly provided by Dr. B. Vogelstein.<br><br> These cell lines were grown in monolayer culture in RPMI 1640 medium or McCoys medium supplemented with glutamine and 5% fetal LY2109761 TGF-beta/Smad 阻害剤 bovine serum at 37 C in a hu midified atmosphere consisting of 5% CO2 and 95% air. Cell survival and apoptotic assays Cells were seeded in 96 well cell culture plates and treated the next day with the given agents. Viable cell numbers were determined using sulforhodamine B assay as described previously. Combination index for drug interaction was calcu lated using the CompuSyn software. Apoptosis was evaluated with an annexin V PE apoptosis detection kit according to the manufacturers instructions. We also detected caspases and PARP cleavage by Western blot analysis as described below as additional indicators of apoptosis. Western blot analysis Preparation of whole cell protein lysates and Western blot analysis were described previously.<br><br> API 1 induces Mcl 1 reduction in API 1 sensitive NSCLC cell lines Human NSCLC cell lines exhibited varied sensitivities to API 1 as evaluated with the SRB assay after a 3 day in cubation. Among them, H1299, H522 and A549 were sensitive to API 1, whereas H226 and H1792 were quite resistant to API 1. Since our previous work investigated the effects of API 1 on the expression of several proteins involved in the extrinsic apoptotic pathway, we focused our current study on determining the effects of API 1 on modulation of the levels of several proteins involved in regulation of the intrinsic apoptotic pathway. In H1299 cells, API 1 decreased the levels of Mcl 1 and survivin at even 2. 5 uM and the levels of Bcl 2 and Bcl XL at 10 uM with no apparent in crease in the levels of the pro apoptotic proteins, Bax, Bad and Bim.