Constructs shRNAlentiviral Tideglusib concentration constructs in pLKO.1 against human LAMP1 was purchased from Sigma Aldrich, and following verification of knockdown, clone ID NM_005561.2-1183s1c1 used to compromise lysosomal integrity. Packaging vectors were obtained through Addgene, Inc. (Cambridge, MA). Lentivirus particles were prepared by transfection of 293 T cells in T75 flasks with 3 μg construct, 2.8 μgpRSV-Rev, 2.4 μgpMDLg/pRRE, and 0.6 μg pMD2.G utilizing FuGENE® 6 Transfection Reagent from F. Hoffmann-La Roche Ltd. (Basel, Switzerland).

Forty-eight and 72 hours following transfection, supernatant was transferred to Bxpc3 cells in the presence of polybrene (8 μg/mL). Transformed cells were selected with puromycin (1 μg/mL) and assayed accordingly. Antibody staining Cells were washed once with PBS prior to fixation with IC

Fixation Buffer (eBiosciences) for 15 minutes at 37°C. Fixed cells were washed with PBS, resuspended in Permeabilization Buffer (eBiosciences), and incubated for 30 minutes at room temperature. Intracellular antigen staining was performed with FITC-antibody buy FHPI dilution of 1:100 in Permeabilization Buffer for 60 minutes at room temperature. Mean fluorescence in FL1 was quantified with a FACSCalibur flow cytometer. Cell buy Selonsertib Viability Cell lines maintained at optimal culture conditions were seeded into 96-well white, clear-bottom plates and following treatment, viability determined with CellTiter-Glo Luminescent Viability Assay from Promega (Madison, WI). Luminescence was quantified with a SpectraMax Gemini microplate spectrofluorometer from Molecular Devices (Silicon Valley, CA). Viability relative to vehicle was fit by non-linear regression and plotted against concentration. Cellular protease

assay Cells were treated in the presence of inhibitors and cytosolic extracts prepared using the digitonin extraction Tryptophan synthase method as previously described [43]. Washed cells were resuspended at 1×106 cells/mL in extraction buffer consisting of sucrose (250 mM), HEPES (20 mM), KCl (10 mM), MgCl2 (1.5 mM), EDTA (1 mM), and digitonin (30 μM). Cells were placed on ice on an orbital shaker for 10 minutes prior to centrifugation for 1 min at 14,000 rpm at 4°C. Supernatants were collected and 20 μL used to detect cleavage of Z-RR-AMC in and equal volume of reaction buffer consisting of sodium acetate (100 mM), NaCl (200 mM), EDTA (4 mM), DTT (10 mM), and Z-RR-AMC (10 μM). Plates were read following incubation at 37 ° for 60 minutes with SpectraMax Gemini microplate spectrofluorometer, Molecular Devices (Silicon Valley, CA) (ex 355 nm, em 450 nm).