At the time point indicated, culture medium was removed and cells were incubated in 2.5 ml/well of 1 mg/ml MTT reagent (dissolved in base maintenance medium lacking phenol red) for 3 h at 37��C in 5% CO2. Suspension cells were collected pre- and post-incubation in MTT by centrifugation. The MTT solution MEK162 molecular weight was then removed and the metabolized MTT reagent was dissolved in isopropanol. Aliquots of the isopropanol-MTT solution were transferred a 96-well microtiter plate in replicate, and absorbances at 570 nm and 690 nm were taken on a SpectraMax M5e instrument (kindly provided by Dr Amar Natarajan, University of Nebraska Medical Center) using SoftMax Pro software (Molecular Devices, Sunnyvale, CA, USA). To determine MTT-specific absorbance, A690 was subtracted from A570.

��-secretase inhibition experiments The cells were seeded at low density in 6-well plates and allowed to adhere for 24 h prior to treatment with ��-secretase inhibitors (signifying 0 h). The inhibitors used were NB2-755, ?281, ?418, ?897 (donated by Novartis, Basel, Switzerland), or ��-secretase inhibitor IV (purchased from Calbiochem/EMD Biosciences). The inhibitors were added at 2 ��M and untreated wells were used as controls. Cells were assayed at 0, 24, 48 and 72 h by the MTT assay (described above) or by mixing aliquots of the cells with 0.4% trypan blue stain (Invitrogen) and counting the cells on a hemocytometer. The percentage of viable cells was calculated as unstained cell number divided by total cell number x 100.

At the 24 h time point, samples of S2-013 cells treated with the Novartis inhibitors were also collected for preparation of cell lysates and western blot analysis (described above). Statistical analysis of data To determine statistical differences in results obtained in the MTT growth assay, the analysis of variance (ANOVA) test was applied with the criterion for significance set at p<0.05. Statistical differences in the results from experiments utilizing the Calbiochem ��-secretase inhibitor were determined by the Student��s t-test and the criterion for significance was set at p<0.05. Results APLP2 is expressed, modified by GAG, and cleaved in human pancreatic cancer cell lines Previously, we found high levels of APLP2 AV-951 expression in several cancer cell lines, including pancreatic cancer lines (S2-013, SUIT2 and Hs766T), prostate cancer lines and a melanoma line (19). We have confirmed the high expression of APLP2 in pancreatic cancer cell lines, using additional pancreatic cancer lines (BxPC3 and Capan-2) and observed additional bands in the molecular mass range previously shown to be GAG-modified APLP2 (20,34,35; Fig. 1A).