Band intensities within equal sized boxes in each lane of the blots for cell lysates and EVs was normalized to the intensity in the respective control (Ctrl) sample.(PDF) pone.0220007.s004.pdf (227K) GUID:?C1F2803B-6C4E-495C-8F24-8F3E3D12F323 S4 Fig: Bafilomycin does not stimulate release of proteins secreted through classical secretion pathway. Lamp1-positive late endosomes.(PDF) pone.0220007.s002.pdf (1.2M) GUID:?A14A8940-83BA-4F6D-8F4D-7366C23261AE S2 Fig: Triton X-100 does not affect nanoluciferase activity in EVs. EVs were isolated from HANL and HANLCD63 expressing TRex293 cells by ultracentrifugation at 100000g for 90 minutes at 4C. The EV pellets were resuspended Isocarboxazid in PBS containing no detergent (-Tx100) or with 0.1%Triton X-100 (+Tx100) and the NLuc luminescence was measured. No difference was obvious between -Tx100 and +Tx100 samples demonstrating that addition of Tx100 to NLuc samples does not affect luminescence.(PDF) pone.0220007.s003.pdf (225K) GUID:?E9046DE2-F6D5-455F-BE08-BF13382C05B2 S3 Fig: Quantitation of the blots in Fig 6B. Band intensities within equal sized boxes in each lane of the blots for cell lysates and EVs was normalized to the intensity in the respective control (Ctrl) sample.(PDF) pone.0220007.s004.pdf (227K) GUID:?C1F2803B-6C4E-495C-8F24-8F3E3D12F323 S4 Fig: Rabbit polyclonal to ZNF658 Bafilomycin does not stimulate release of proteins secreted through classical secretion pathway. Extracellular Nluc activity was measured in HANLCD63 and SecNL for 2h under control (Ctrl; blue lines) or after addition of bafilomycin (Baf; red lines). While extracellular release of HANLCD63 was enhanced by bafilomycin, secretion Isocarboxazid of SecNL was greatly inhibited.(PDF) pone.0220007.s005.pdf (237K) GUID:?CB885607-C9AF-4F63-A33F-A9F78E1A8439 S5 Fig: Ammonium chloride does not affect bafilomycin-stimulated EV secretion. NLuc luminescence was measured in conditioned culture media of HANL and HANLCD63 cells treated without (-Baf) or with 200nM bafilomycin (+Baf) and were either not co-treated (-AmmCl) or co-treated with 10mM ammonium chloride (+AmmCl) as an alkalizing agent. Isocarboxazid No difference was observed in +Baf samples with or without ammonium chloride cotreatment. This result shows that alkalizing agents do not influence increased EV release due to V-ATPase inhibitors.(PDF) pone.0220007.s006.pdf (231K) GUID:?91F217B3-A654-403F-9965-912C7E85C7D1 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Extracellular vesicles (EVs) are thought to be important in cell-cell communication and have elicited extraordinary interest as potential biomarkers of disease. However, quantitative methods to enable elucidation of mechanisms underlying release are few. Here, we describe a cell-based assay for monitoring EV release using the EV-enriched tetraspanin CD63 fused to the small, ATP-independent reporter enzyme, Nanoluciferase. Release of CD63-containing EVs from stably expressing cell lines was monitored by comparing luciferase activity in culture media to that remaining in cells. HEK293, U2OS, U87 and SKMel28 cells released 0.3%-0.6% of total cellular CD63 in the form of EVs over 5 hrs, varying by cell line. To identify cellular machinery important for secretion of CD63-containing EVs, we performed a screen of biologically active chemicals in HEK293 cells. While a majority of compounds did not significantly affect EV release, treating cells with the plecomacrolides bafilomycin or concanamycin, known to inhibit the V-ATPase, dramatically increased EV release. Isocarboxazid Interestingly, alkalization of the endosomal lumen using weak bases had no effect, suggesting a pH-independent enhancement of EV release by V-ATPase inhibitors. The ability to quantify EVs in small samples will enable future detailed studies of release kinetics as well as further chemical and genetic screening to define pathways involved in EV secretion. Introduction Extracellular vesicles (EVs) are released by cells and found in most biological fluids including urine, plasma, cerebrospinal fluid, saliva etc. as well as in tissue culture conditioned media. EVs are thought to mediate cell-cell communication  and may carry a variety of proteins, lipids and RNA with potential to impact target cell physiology. It has been proposed that EVs modulate tumor environments to allow for tumor seeding and growth and promote angiogenesis [2C8]. EVs have also been implicated in the prion-like spread of neuropathogenic protein aggregates in several neurodegenerative diseases [9C15]. Certain Isocarboxazid viruses and bacteria such as hepatitis A virus , herpesvirus 6 , HTLV-1 , HIV [19,20] and uropathogenic [21, 22] may use the cellular pathways of EV biogenesis for extracellular release. Recent studies in many laboratories have focused on exploring the utility of EVs isolated directly from biological fluids as disease biomarkers [23,24]. Finally, EVs are also being developed as therapeutic agents capable of delivering drugs to specific tissues or organs in the body [3,25,26]. EVs are produced in at least two distinct.