Supplementary MaterialsS1 Fig: Characterisation of exosomes secreted from KLEC. was performed using the KSHV-miR LNA PCR primer models (Exiqon). In all panels, except to panel B, the graphs present the mean and standard deviation of 3 biological repeats.(TIF) ppat.1006524.s001.tif (1.0M) GUID:?9466E34F-7714-45EB-A0A5-C7B8A71563E2 S2 Fig: KLEC-derived exosomes are being taken up by na?ve cells. LEC were incubated with fluorescently labelled exosomes and analysed using a fluorescence-activated cell sorter (FACS).(TIF) ppat.1006524.s002.tif (198K) GUID:?E1CB626B-92E3-4390-8D01-75921E15EB57 S3 Fig: KLEC-derived exosomes induce the reverse Warburg effect. (A) LEC were educated using the indicated number of exosomes collected from KLEC growth media and analysed using the Seahorse XF24 Analyser for oxygen consumption rate. The bar graph presents the average base line oxygen consumption rate. (B) Oxygen consumption rate of uneducated LEC, and LEC and KLEC co-cultured in transwell plates. (C) The indicated metabolites concentrations as measured in educated cells using CE-TOFMS and CE-QqQMS (Human Metabolome Technologies, Inc.). (D) LEC were educated using KLEC-derived exosomes, then grown for additional 5 days in exosome free media and analysed using the Seahorse XF24 Analyser for oxygen consumption rate. (E-F) HUVEC were educated using the indicated exosomes and analysed for oxygen consumption rate using the Seahorse XF24 Analyser (E) or for mitochondria volume (F) as previously described . The bar graph presents the average mitochondrial volume in cells (Mean+SD, n = 3).(TIF) ppat.1006524.s003.tif (438K) GUID:?205A3431-3819-4A49-B79A-9CDF57F6B15C S4 Fig: Characterisation of exosomes secreted from AKATA cells. (A) Lysates from purified exosomes or EBV (10g) were separated by SDS/PAGE and analysed by immunoblot for the viral protein gp125. (B) Lysates from purified exosomes or EBV (10g) were separated by SDS/PAGE and analysed by immunoblot for expression of the exosomal Benzyl chloroformate marker ALIX.(TIF) ppat.1006524.s004.tif (91K) GUID:?533AC5A1-9DFB-4A8D-8618-DDDF40E9DA1F S5 Fig: miR-210 is transfer in exosomes to induce reverse Warburg effect. (A) Levels of miR210 in exosomes secreted from 293T or HCT-116 force expressing miR210. Detection of mature hsa-miR-210 was performed using a specific LNA PCR primer set (Exiqon). (B) Expression levels of ISCU1 in cells educated using miR-210 exosomes. mRNA amounts had been dependant on quantitative real-time PCR (qRT-PCR). Tubulin beta (TUBB) amounts had been useful for normalisation. (C) Air consumption price (OCR) as assessed using the Seahorse XF24 Analyser. Cells had been seeded at a denseness of 4×104 cells per Trdn well as well as the assay was performed based on the producers Mito stress process.(TIF) ppat.1006524.s005.tif (235K) GUID:?BBBBEE0C-2822-40AF-97B0-A15D9EE856C1 S6 Fig: KLEC more than express Benzyl chloroformate the monocarboxylate transporters MCT 1 Benzyl chloroformate and 2. mRNA amounts had been dependant on quantitative real-time PCR (qRT-PCR). Tubulin beta (TUBB) amounts had been useful for normalisation.(TIF) ppat.1006524.s006.tif (98K) GUID:?E8E2A67D-BA96-4664-BFD6-27AB22D83FC0 S1 Desk: Expression degrees of the KSHV miRNAs in KLEC and KLEC-derived exosomes. The expression level was calculated as fraction of total reads detected in KLEC-derived and KLEC exosomes.(TIF) ppat.1006524.s007.tif (1.4M) GUID:?4B2504EE-32CF-4F2E-9D6A-8E0FA79B45DB S2 Desk: Relative manifestation degrees of selected miRNAs in KLEC in comparison to LEC. (TIF) ppat.1006524.s008.tif (975K) GUID:?D8F23303-217C-44B2-B66F-34E6A48FCBA7 S3 Desk: Comparative expression degrees of decided on miRNAs in KLEC derived exosomes in comparison to LEC derived exosomes. (TIF) ppat.1006524.s009.tif (747K) GUID:?6035C865-DC9E-47EE-BFC6-E0068F630573 Data Availability StatementAll relevant data are inside the paper and its own Supporting Information documents. Abstract Metabolic adjustments inside the cell and its own niche influence cell fate and are involved in many diseases and disorders including cancer and viral infections. Kaposis sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposis sarcoma (KS). KSHV latently infected cells express only a subset of viral genes, mainly located within the latency-associated region, among them 12 microRNAs. Notably, these miRNAs are responsible for inducing the Warburg effect in infected cells. Here we identify a novel mechanism enabling KSHV to manipulate the metabolic nature of the tumour microenvironment. We demonstrate that KSHV infected cells specifically transfer the virus-encoded microRNAs to surrounding cells via exosomes. This flow of genetic information results in a metabolic shift toward aerobic glycolysis in the surrounding noninfected cells. Importantly, this exosome-mediated metabolic reprogramming of neighbouring cells supports the growth of infected cells, thereby contributing to viral fitness. Finally, our data show that this miRNA transfer-based legislation of cell fat burning capacity is an over-all system used by various other herpesviruses, such as for example EBV, aswell for the transfer of nonviral onco-miRs. This exosome-based crosstalk provides infections with a system for noninfectious transfer of hereditary material without creation.