The retroviral vector was con structed as follows human p8 cDNA was subcloned in HindIII and XhoI restriction sites of the pLPC plasmid in the antisense orientation. Amphotrope human p8 expressing retrovirus was then generated by transient transfection using Phoenix amphotrope packaging cells. Viral supernatant was used to infect Panc 1 and BxPc 3 pancreatic except cells and the population of p8 silenced cells was isolated by selection in presence of puromycin. As control, cells were infected with the pLPC empty vector. BxPc 3 rasV12 expressing Inhibitors,Modulators,Libraries cells pLPC rasV12 and pLPC plasmids were obtained from S. Lowe. Phoenix amphotrope packaging cells were plated in a 6 well plate, incubated for 24 hours, Inhibitors,Modulators,Libraries then transfected with Inhibitors,Modulators,Libraries PEI with 5g of retroviral plasmid. After 48 hours, the medium containing virus was filtered to obtain the viral supernatant.

Tar get BxPc 3 were plated at 2 105 cells per 35 mm dish and incubated overnight. For infections, the culture medium was replaced by an appropriate mix of the viral superna tant and culture medium, supplemented with 4g/ ml polybrene, and cells were incubated at 37 C. BxPc 3 rasV12 expressing cells were selected with puromycin. Cells infected with the pLPC empty vector were used as control. Inhibitors,Modulators,Libraries Western blot analyses One hundredg of total protein extracted from cells was separated with standard procedures on 15. 0% SDS PAGE using the Inhibitors,Modulators,Libraries Mini Protean System and transferred to a nitrocellulose membrane. The intracellular level of p8 was estimated by Western blot using a polyclo nal antibody raised against human p8.

Growth curves One hundred thousand cells per well were plated in a series of 35 mm culture dishes. The cell number was esti mated daily in triplicate, during 1 to 5 days, in a haemocytometer. Within experiments, each point was determined at least two times. Cell transfection and gene reporter assays Panc 1 and BxPc 3 were cultivated in 30 mm diam eter culture dishes for 24 hours Cisplatin buy then transiently transfected with 0. 5g of p8 CAT reporter plasmid and 0. 5g of pCMV gal plasmid using the Fugene reagent in accordance with the manufacturers protocol. The p8 CAT plasmid is the previously reported p 1471 37p8 CAT promoter construct. Reporter activi ties were measured as previously described. Briefly, cell extracts were prepared with the reporter lysis buffer 24 hours after transfection and CAT activity was determined by the phase extraction procedure and galactosidase assay was performed essentially as described in Sambrook et al. CAT activity was nor malized to galactosidase activity. Experiments were carried out in triplicate and repeated at least two times. Expression plasmids were co transfected with p8 CAT and pCMV gal plasmids as indicated.