Tumor cell enrichment from ascites Tumor cell enrichment was based on the expression of CD326, a human epithelial antigen also known as EpCAM, which is broadly expressed on cells of epithelial origin and derived tumor cells. CD326 cells were positively sellectchem selected using autoMACs Inhibitors,Modulators,Libraries columns from ascites collected from patients diagnosed with EOC. The percentage of CD326 cells in the positive fraction was more than 80% as assessed using a FACScan flow cytometer. Cell culture and reagents BG 1 cells derived from a solid tumor tissue of a patient with stage III ovarian adenocarcinoma were maintained in Dulbeccos modified minimum essential medium supplemented with 10% fetal bovine serum, 2 mmol/L L glutamine, 0. 1 mg/mL strepto mycin and 100 U/mL penicillin.

The Inhibitors,Modulators,Libraries human ovarian carcinoma cell lines SKOV 3 and OVCAR 3 were maintained in RPMI 1640 medium containing Inhibitors,Modulators,Libraries 0. 1 mg/mL streptomycin, 100 U/mL penicillin, 2 mmol/L L glutamine and 10% FBS. Triciribine was purchased from Calbiochem. Generation of BG 1 clones stably overexpressing GILZ BG 1 cells were transfected using jetPEI according to the manufacturers protocol, with the GILZ encoding vector pcDNA3 GILZ or with the empty vector pcDNA3 as a control. Forty eight hours after transfection, stably transfected cells were selected by a 2 week treatment with 500g/mL of Geneticin and cloned by limiting dilution. Clones were then screened for GILZ expression using quantitative real time PCR and immunoblot assays. GILZ expression remained stable over 7 days in culture. We thus generated BG 1 clones that stably and strongly expressed GILZ, named pGILZ clones.

BG 1 clones stably transfected with an empty vector, named CTRL clones, were used as con trol. GILZ silencing Small interfering RNA duplexes were synthe sized and tested for specific inhibition of GILZ expression as described previously. BG 1 cells were transfected with 4g/well GILZ siRNA Inhibitors,Modulators,Libraries or control siRNA, purchased from Qiagen, by the lipofectamine method using X tremeGENE siRNA Inhibitors,Modulators,Libraries transfection reagent according to the manufacturers instructions. Transfection efficiency was between 80% and 90%, as assessed by using fluorescent random siRNA siRNA AlexaFluor 488. RT PCR procedures Total RNA was extracted using a RNeasy Mini kit. The RNA was transcribed into cDNA by reverse tran scription http://www.selleckchem.com/products/Bortezomib.html with random hexamers and Moloney murine leukemia virus reverse tran scriptase. GILZ mRNA was quantified by real time PCR on a Light Cycler instrument using the FastStart DNA Master SYBER Green kit as described previously. Val ues were normalized to those for actin mRNA and are thymidine uptake Cells were seeded in triplicate on 96 well plates at a den sity of 1 104 cells/well in DMEM medium with 10% FBS.