vFLIPs (19C21) However subsequent work by our laboratory and others showed that K13 does not act as a vFLIP but instead directly interacts with the NEMO/IKK  subunit of the Androgen Receptor Antagonists IKK complex to selectively activate the NF B pathway (22C24) In this study we have taken advantage of this unique ability of K13 to selectively activate the NF B pathway and used it as a molecular tool to study the role of the NF B pathway in IL6- independent growth of murine plasmacytoma cells MATERIALS AND METHODS Cell lines and ReagentsªT1165 and B9 cells were grown in RPMI medium supplemented with 10% (v/v) FCS 100 units/ml penicillin 100  g/ml streptomycin 1 mM sodium pyruvate 2 mM glutamine (all from Invitrogen) and 10 ng/ml and 5 ng/ml of recombinant human IL6 (PeproTech Inc) respectively HEK-293FT cells (Invitrogen) were grown in.

Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum 100 units/ml penicillin 100  g/ml streptomycin and 250  g/ml Geneticin sulfate in a humidified atmosphere containing 5% CO2 at 37 ãC Dexamethasone and As2O3 (arsenic trioxide) were purchased from Sigma Bay-11-7082 and INCB018424 were purchased from Tocris (Ellisville MO) and ChemieTek (Indianapolis IN) respectively Retrovirus Lentivirus MK-4827 Constructs and Virus InfectionªRetrovirus constructs containing C-terminal FLAG epitopetagged wild-type and mutant vFLIP K13 and E8 were generated in murine stem cell virus (MSCV) neo-based retroviral vector and amphotropic viruses were generated and used for infection as described previously (23) Lentivirus constructs encoding C-terminal FLAG-tagged wild-type .

Tax and its mutants (M22 and M47) were generated in pLENTI6/V5-based vector Bacterial infection (Invitrogen) A retroviral vector expressing the firefly luciferase gene was constructed in the pRetroQ-RSV vector (Clontech) in which the CMV promoter had been replaced with an RSV promoter The MSCV-Bcl-2-IRES-GFP and MSCV-Bcl-xL-IRESGFP constructs were kindly provided by Dr Emily Cheng (Human Oncology and Pathogenesis Program Memorial Sloan-Kettering Cancer Center New York NY) The MSCVpuro- WT-Mcl-1 construct was a kind gift from Dr Opferman (Department of Biochemistry St Jude Children’s Research Hospital Memphis TN) Recombinant retroviruses and lentiviruses were generated in the HEK293-FT cells as described previously and used to infect T1165 and B9 cells (25 26) All mined using the IVIS200 system (Caliper Hopkinton MA) At autopsy cells were harvested from an abdominal plasmacytoma and the spleen of a mouse was injected with T1165-Luc-K13IL6 cells and cultured in RPMI medium with 10% FCS in the absence of IL6 In parallel cells were harvested from the spleen of an animal injected with the T1165-Luc-vector cells .