thasone and As 2 O 3 (arsenic trioxide) were purchased from Sigma. Bay-11-7082 and INCB018424 were purchased from Tocris (Ellisville, MO) and ChemieTek (Indianapolis, IN), respectively. Retrovirus, Lentivirus Constructs, and Virus Infection —Ret- rovirus leurocristine constructs containing C-terminal FLAG epitope- tagged 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 (Invit- rogen). 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 pro- moter. The MSCV-Bcl-2-IRES-GFP and MSCV-Bcl-xL-IRES- GFP constructs were kindly provided by Dr. Emily Cheng (Human Oncology and Pathogenesis Program, Memorial leurocristine 2068-78-2 (Sigma,:50,000), Mcl-1 (Santa Cruz Biotechnology, Inc., sc-19,:1000), Bcl-2 (Santa Cruz Biotechnology, Inc., sc-492,:000), Bcl-xL (Santa Cruz Biotechnology, Inc., sc-634,:1000), tubulin (Sigma,:50,000), cleaved caspase 3 (Cell Signaling Technology, Inc., 8G10,:1000), poly(ADP-ribose) polymerase (PARP) (Cell Signaling Technology, Inc., 9542,:1000), I B- (Santa Cruz Biotechnology, Inc., sc-864,:1000), p-I B (Ser- 32, 9241, Cell Signaling Technology, Inc.,:1000), Akt (Cell Signaling Technology, Inc.

9272,:1000), phospho-Akt (Cell Signaling Technology, Inc., Ser-473, 9271,:1000), stress-acti- vated protein kinase (SAPK)/JNK (Cell Signaling Technology, Inc., 9252,:1000), and phospho-SAPK/JNK (Cell Signaling Technology, Inc., 9251,:1000). Animal Experiments —T1165 vector and T1165-K13 IL6 cells were transduced with a pRetroQ-RSV-Luc retroviral vec- tor that expresses the firefly gene under an RSV promoter, and infected cells were selected with puromycin. Subsequently, 4- to 6-week-old buy leurocristine BALB/cAnNCr mice (Charles River Laborato- ries, Wilmington, MA) were inoculated intraperitoneally with Sloan-Kettering Cancer Center, New York, NY). The MSCV- 0 0 7 cells. Tumor growth in the peritoneum was monitored puro-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 lenti- viruses were generated in the HEK293-FT cells as described previously and used to infect T1165 and B9 cells (25, 26). All weekly by physical examination and bioluminescence imaging for0 weeks. For bioluminescence imaging, after mild anesthesia with isoflurane, animals were injected intraperitoneally with an radioactive aqueous solution of D-luciferin (Biosynth, Naperville, IL) at50 g/g body weight in PBS, and firefly luciferase activity was deter- AUGUST2, 2011 • VOLUME 286 • NUMBER 32 JOURNAL OF BIOLOGICAL CHEMISTRY 27989 Downloaded from www.jbc.org at NYU School of Medicine Library, on March 7, 2012 2 NF- B Confers IL6 Independence FIGURE. K13 protects the T1165 murine plasmacytoma cell line against IL6 withdrawal-induced apoptosis. A , expression of FLAG-K13 in T1165 cells as revealed by Western blotting ( IB ) with a FLAG antibody. B and C , T1165 cells expressing an empty vector or K13 were grown in triplicate in a 96-well plate in the presence or absence of IL6, and cell viability was measured 48 h later using an MTS assay. The values shown are mean S.D. of two independent experiments performed in triplicate.

p 0.05 as compared with vector cells ( B ). Cells were stained with SYTOX Green, a cell-impermeable nuclear dye that stains the nuclei of dead cells, and were examined under a fluorescence microscope or under phase-contrast microscope and photographed ( C ). D , DNA content analysis shows significant increase in sub-G 0 /G fraction in T1165-vector cells upon withdrawal of IL6, which was absent in K13-expressing cells. mined using the IVIS200 system (Caliper, Hopkinton, MA). At a