, St. Louis, MO) at a final concentration of 8 mM. Asynchronous cells were seeded 24 hours before treatment with 11a, such that cells were approximately 50%-60% confluent at the time of 11a addition. The final concentration of 11a in nM – ��M range was achieved by diluting 11a in the fresh medium, and 0.1% MG132 DMSO DMSO was used as the control for each experiment. All-trans retinoic acid, doxorubicin, etoposide, staurosporine and 3-aminobenzamide were purchased from Sigma (St. Louis, MO). For cell cycle analysis, cells were serum-starved for 24 hours in order to achieve G0 synchronization. The cells were then allowed to re-enter the cell cycle by supplementing with DMEM plus 10% FBS containing the indicated concentrations of 11a.

Retrovirus packaging, infection and stable cell line generation The packaging plasmids pME-VSVG, pHIT60 and pLNCX were purchased from OpenBiosystems (Huntsville, AL). Retroviruses were packaged in HEK293T cells transfected with 3.8 ��g pME-VSVG, 1.4 ��g pHIT60 and 3.8 ��g pLNCX-GFP or pLNCX-PNR using transIT-LT1 reagent (Mirus Bio) according to the manufacturer��s instructions. Six hours after transfection, the medium was changed. The virus particles were then harvested 24 to 48 hours later using a 0.45 ��m syringe filter (Thermo Scientific). To infect the cells with retroviruses, the viruses were mixed with an equal volume of fresh media supplemented with 10% FBS. Polybrene was added at a final concentration of 5 ��g/mL in order to increase the infection efficiency. The medium was changed 6 hours after infection.

Cells were selected with G418 (800 ��g/ml) for a week to generate stable cell lines expressing GFP or PNR. CellTiter Glo luminescent cell viability assays One thousand cells per well were seeded in quadruplicate in a 384-well plate and treated with the indicated concentrations of 11a for a week. Cells were then subjected to the CellTiter Glo luminescent cell viability assay according to the manufacturer��s instructions (Promega, Madison, WI). The IC50 values were calculated by using the XLfitTM add-in for Excel. Luciferase reporter assays The DR2-driven luciferase reporter, TLX and COUP-TFI plasmids were kind gifts from Dr. Ronald Evans. COUP-TFII plasmid was a kind gift from Dr. Michael Gould. The other plasmids were purchased from OpenBiosystems (Huntsville, AL). Luciferase assays were performed using the Luciferase Assay System (Promega, Madison, WI).

HEK293T cells were seeded in a 96-well plate (2 �� 103/well). After 24 hours, cells were transfected using transIT-LT1 (Mirus Bio) with 20ng DR2-driven luciferase reporter, 10 ng ��-galactosidase reporter, and 20ng CMV expression vector for control, PNR, TLX, COUP-TFI or COUP-TFII. Compound 11a was added 24 hours Cilengitide after transfection, and luciferase activity was determined after incubation for an additional 24 hours. ��-galactosidase activity was used to normalize for transfection efficiency.