Fluorescence intensity (max 529 nM) was quantified in the FL1 channel with a FACSCalibur flow cytometer. Caspase-3 activity Cells were maintained at optimal conditions and seeded in 96-well black-bottom plates in a volume see more of 100 μL. Following treatment, 5X assay buffer containing EDTA (10 mM), CHAPS (5 %), HEPES (100 mM), DTT (25 mM), and Ac-DEVD-AMC (250 μM) was added directly to the cell media and incubated for two hours at 37°C on a microplate shaker, and liberated AMC quantified with a SpectraMax Gemini

microplate spectrofluorometer, Molecular Devices (ex 355 nm, em 450 nm). Caspase-3 activity is normalized to the absence of inhibitor. Statistical analysis Statistical analysis and data plotting was conducted

using GraphPad Prism check details (GraphPad Software, San Diego, CA). Data represents the mean ± SEM. Viability IC50 values at 18 hours were calculated by line fitting normalized viability versus concentration with non-linear regression and statistical significance determined using one-way ANOVA. Differences in viability, caspase-3 activity, apoptosis, and oxidation status were analyzed using two-way ANOVA to identify differences and confirmed with paired two-tailed t-tests. Blood cytology and biochemistry SB202190 supplier results were analyzed using one-way ANOVA with Tukey’s multiple comparison test. Statistical analysis for the difference in tumor volume between treatments groups was determined with the repeated measures ANOVA. Kaplan-Meier survival curves were plotted and differences compared with a log-rank test. A p-value of less than 0.05 was L-gulonolactone oxidase considered significant for all tests. Acknowledgements This work was funded by a grant from the American Cancer Society [MRSG08019-01CDD] (WGH), a Veteran’s Administration Merit Award [1136919] (WGH), and a Surgical Oncology Training Grant [5T32CA009621-22] (JRH). The authors would like to give appreciation to Brian Belt, Stacy Suess, and Jesse Gibbs for

their technical support and assistance in experiments. Electronic supplementary material Additional file 1: Figure S1. In vivo efficacy of sigma-2 receptor ligands. Female C57BL/6 mice inoculated subcutaneously with 1×106 Panco2 cells were treated daily with sigma-2 receptor ligands when tumors reached an average of 5 mm in diameter. Data represents mean ± SEM, n = 7–10 per group. Mice received daily treatment through the duration presented. (TIFF 4 MB) Additional file 2: Figure S2. Colocalization of SW120 and PB385 in Bxpc3 and Aspc1 pancreatic cancer cell lines by fluorescence microscopy. Live cells were imaged following incubated with LysoTracker Red (50 nM), red, and fluorescent sigma-2 receptor ligand (500 μM), green, for 30 minutes at 37°C prior to nucleic acid counterstaining with Hoechst, blue, scale bar = 20 μm. (JPEG 8 MB) Additional file 3: Figure S3.