Better understanding of the susceptibility of cancers to certain death pathways will ultimately allow tailoring of sigma-2 receptor ligand treatment inhibitor Veliparib choice. Materials and Methods Cell Culture Cell lines were maintained in RPMI media (GIBCO) supplemented with L-glutamine (2mM), (HEPES) (1mM), pyruvate (1mM), sodium bicarbonate (0.075%w/v), penicillin and streptomycin (100IU/mL), amphotericin (0.25��g/mL), and 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA). Cells were seeded at a density of 2 x 105/mL unless otherwise stated and maintained in a humidified atmosphere of 5% CO2 at 37��C. Compounds Sigma-2 receptor ligands were synthesized as previously described [10,16,17,40-42].

The imaging dyes acridine orange and LysoTracker Red were obtained from Invitrogen (Carlsbad, CA), FITC mouse anti-human CD107a (LAMP1) and CD107b (LAMP2) antibodies from BD Biosciences (Franklin Lakes, NJ), peptidase inhibitors CA-074-Me and pepstatin A, and fluorogenic peptidase substrate Z-RR-AMC from Enzo Life Sciences (Plymouth Meeting, PA), caspase-3 inhibitor Z-DEVD-FMK from R&D Systems (Minneapolis, MN); caspase-3 substrate Ac-DEVD-AMC from Bachem Biosciences, Inc (King of Prussia, PA); All other reagents were obtained from Sigma-Alrich (St. Louis, MO) unless otherwise stated. Compounds were dissolved in DMSO with final concentrations less than 0.3%. In vivo tumor treatment Athymic nude mice from Harlan Bioproducts, Inc. were inoculated subcutaneously with 1×106 Bxpc3 cells in the right flank.

Tumor sizes were monitored with calipers and when tumors reached an average of 5mm in diameter, mice were randomized and treated daily with equimolar doses of sigma-2 receptor ligands SV119 (1.0mg), SW43 (1.1mg), PB28 (0.9mg), or PB282 (0.9mg) resuspended in vehicle consisting of 5% DMSO, 5% EtOH, and 10% Cremophor in 1X PBS and injected intraperitoneally. Data represents mean��SEM, n=7�C10 per group. Confocal microscopy Cells grown on glass cover slips were incubated with SW120 or PB385 (100 nM) in the presence of LysoTracker Red (25 nM) for 30 minutes at 37��C. Cells were washed with PBS and fixed in 2% paraformaldehyde for 30 minutes at 37��C prior to additional washing and mounting with ProLong Gold antifade reagent. Confocal imaging was performed on a Carl Zeiss Axiovert 100 inverted microscope, fitted with LSM 510 laser scanning microscope camera and software. Images were collected with filter bandwidths corresponding to 505�C530nm for green, 560�C615nm for red, and>650nm for far red, with 4 scans over 11.8 seconds. Fluorescence microscopy Cells grown on glass cover slips were loaded with acridine orange (2��g/mL) Entinostat for 15 minutes at 37��C prior to treatment for one hour with compounds.