Halt protease inhibitor

Soluble protein extraction from HEK293T cells was performed using Cytobuster Protein Extraction Reagent (Fisher Scientific) according to the manufacturer’s instructions. Protein extraction solutions were supplemented with 1X Halt Protease Inhibitor (Fisher Scientific) and 1X Halt Phosphatase Inhibitor (Fisher Scientific) Cocktails. Protein concentrations were determined using A280 on a NanoDrop 1,000 Spectrophotometer and subsequently analyzed by Western immunoblotting. Equivalent amounts of protein (40 µg) were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore Sigma) using the Trans-Blot Semi-Dry Electrophoretic Transfer Cell System (Bio-Rad); Ponceau S staining was used to assess the equivalency of protein loading after SDS-PAGE transfer. Membranes were washed with 1.0M Tris-buffered saline (TBS), pH 7.4, blocked with 5% Bovine Serum Albumin (BSA) in TBS for 1 hour at room temperature, and then incubated with primary antibodies (Key Resources Table) in 5% BSA in TBS-T (0.1% Tween 20) overnight at 4°C. Following TBS-T washes, membranes were incubated with peroxidase-conjugated secondary antibodies in TBS-T (Key Resources Table) and protein bands were detected using Clarity Western ECL Substrate (Bio-Rad) and visualized using the ChemiDoc Imaging System (Bio-Rad).

Frozen tissue samples were embedded in Cryoembedding Medium (Medite, Germany) and cut into 20 μm sections using a CryoStar NX70 cryostat (Fisher Scientific, Schwerte). For protein analysis by ELISA, tissue slices were incubated on ice for 30–60 min in 1xLM lysis buffer from NADH dehydrogenase (Complex I) Human SimpleStep^TM ELISA kit (Abcam, ab178011). Buffers were added in complete mini Protease Inhibitor Cocktail (Roche, Germany), HALT Protease inhibitor (Fisher Scientific, Germany) and Phosphatase inhibitor cocktails 2 and 3 (Sigma, Germany). Lysates were vortexed before centrifugation for 10 min at 13,000 × g to precipitate tissue debris. Supernatants were aliquoted and stored at −80 °C until analysis. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay (Sigma, Germany).

Seminal PSA was purchased from Calbiochem (San Diego, CA). The WHO International Standard PSA Free (NIBSC code 96/668) was obtained from National Institute for Biological Standards and Control (Herts, UK). Seminal PSAs were purified from seminal plasma of healthy men and were used as normal control. Synthetic peptides IRNKS and IRDKS were obtained from BEX Co. Ltd. (Tokyo). Bovine serum albumin (BSA) and phenylmethylsulfonyl fluoride (PMSF) were purchased from Merck (Darmstadt, Germany) and Halt^TM protease inhibitor was obtained from Thermo Fisher Scientific (Rockford, IL).

HTM cells (N17TM-2, N27TM-2 and N27TM-6) were plated into 96 well plates at 15,000 cells per well. One week after the cells reached confluency media was replaced with media containing 1% FBS and 2ng/ml TGFβ2 with and without 2μM FUD. Two days later the cells were processed for OCW analysis as previously described [19 (link)]. Briefly, cells were extracted with a hypotonic buffer (20mM HEPES, pH 7.4, 1mM EDTA, 1% sodium deoxycholate and HALT^TM protease inhibitor (Thermo Fisher Scientific)) leaving behind insoluble extracellular matrix (ECM). The DOC insoluble ECM was fixed with 4% paraformaldehyde and the total protein in the wells was labeled with IRDye 680 NHS ester (LI-COR). Wells were then blocked and fibronectin was detected using rabbit anti-fibronectin sera followed by the IRDye 800CW-conjugated goat anti-rabbit IgG (LI-COR). The plates were then read on a LI-COR Odyssey CLx scanner and analyzed using the LI-COR Image Studio v. 5.0.21 software. The antibody signal was normalized to the corresponding NHS ester signal (total protein).

Desulfovibrio sp. strain DF1 and E. coli were grown anaerobically in a carbonate-buffered minimal medium reduced with 1 mM Titanuim(III)nitriloacetate (Ti(III)-NTA) (basal medium, Widdel and Pfennig, 1981 (link); trace elements solution, Widdel et al., 1983 (link); Ti(III)NTA solution, Moench and Zeikus, 1983 (link); selenium-tungstate solution, Tschech and Pfennig, 1984 (link); vitamin solution, Pfennig, 1978 (link)) and 12 mM SQ or 6 mM glucose (E. coli) or 10 mM DHPS (Desulfovibrio sp.). For sulfate-respiring growth conditions, Desulfovibrio sp. DF1 was grown with 10 mM lactate as the carbon source and electron donor and 20 mM sulfate as the terminal electron acceptor. The gas phase was 20% CO₂ and 80% N₂. All cultures were incubated at 30°C. Cultures for proteomic analysis, 2D-PAGE and enzyme assays in cell-free extracts were harvested in late exponential phase at an optical density (OD₅₈₀) of about 0.4. The cells were resuspended in 50 mM Tris–HCl buffer, pH 8.0, containing 5 mM MgCl₂, 1.7 μg/ml DNAse I and 1× Halt^TM protease inhibitor (ThermoFisher Scientific) and disrupted in a cooled French pressure cell.
E. coli for cloning and overexpression was grown in LB medium with, if necessary, 30 μg/ml kanamycin, 100 μg/ml ampicillin or 35 μg/ml chloramphenicol, aerobically on an orbital shaker at 37 or 15°C. For details on the cloning, see below.

NCI-60 and MCF10a EVs were processed as previously described in great detail [40 (link),43 (link)]. The enrichment efficacy and purity of samples has been demonstrated numerous times through nanoparticle tracking, immunoblot analysis, and electron microscopy by our laboratory [43 (link),44 (link),45 (link),46 (link),47 (link)]. Briefly, serum-free cell-conditioned medium was aspirated from cell culture plates in three biological replicates, and centrifuged at 500× g for 5 min, then at 2000× g for 30 min before incubation overnight with a 1:1 volume of 2X PEG solution (16% (w/v) polyethylene glycol, 1 M NaCl). Following the polyethylene glycol incubation, samples were centrifuged at 3214× g for 60 min, and pellets were resuspended in PBS for an ultracentrifugation purification step (100,000× g for 70 min). Final pellets were lysed in strong lysis buffer (5% SDS, 10 mM EDTA, 120 mM Tris-HCl pH 6.8, 2.5% β-mercaptoethanol, 8 M urea) with the Halt^tm protease inhibitor (ThermoFisher, Waltham, MA, USA, 78438) added. Vesicle protein quantification was performed using the fluorescence-based EZQ™ Kit (ThermoFisher, Waltham, MA, USA, R33200) according to the manufacturer’s instructions.