Sequence Coverage (%) Profilin-1 P UD 2

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1 Protein Name Accession Number (Swissprot) Sequence Coverage (%) No. of MS/MS Queries Mascot Score 1 Reference Cytoskeletal proteins Beta-actin P Alpha-actin P Beta-actin-like protein 2 Q562R Profilin-1 P UD 2 Adhesion Integrin-beta-1 P UD Integrin-alpha-3 P UD Integrin-alpha-5 P UD CD44 P Galectin-1 P Enzymes Glyceraldehyde-3-phosphate P UD dehydrogenase Alpha-enolase P Beta-enolase P Fructose-bisphosphate P UD aldolase A Pyruvate kinase P UD L-lactate dehydrogenase B P UD chain 5 -nucleotidase P UD Sodium/potassiumtransporting P ATPase subunit alpha-1 Sodium/potassiumtransporting ATPase subunit beta-3 P UD Signalng proteins G(i) alpha-3 P UD G(i) alpha-2 P UD G-protein beta-1 P UD G-protein beta-4 Q9HAV G-protein beta-2 P P Prostaglandin F2 receptor negative regulator Q9P2B Membrane transport and fusion Rab-15 P UD Rab-35 Q UD Annexin A2 P UD Myristoylated alanine-rich protein kinase C substrate P UD 20

2 Protein Name Accession Number (Swissprot) Sequence Coverage (%) No. of MS/MS Queries Mascot Score 1 (MARCKS) ADP-ribosylation factor 4 P Reference 47 Tetraspannins CD63 P UD CD151 P Others Collagen alpha-3 P UD Collagen alpha-1 P UD Collagen alpha-2 P Solute carrier family 16, P member 1 Basigin/CD147 P Elongation factor 1-alpha P UD Brain abundant, membrane P UD attached signal protein 1 Solute carrier family 29 Q Score based on Mascot assignment, and is a statistical measure of accuracy of assignation, a higher score implies greater confidence of protein identity 2 UD: Urinary Exosome Protein Database ( Table S1. Proteomic analysis of U87 exosomal proteins by mass spectroscopy 100μg of U87 exosomes were extracted by chloroform/methanol precipitation and subjected to mass spectroscopy analysis as described in the methods. MS/MS spectra (peak lists) were searched against the SwissProt database using Mascot version 2.2 (Matrixscience, London, UK) and the following parameters: peptide tolerance 2.5Da, 13 C=0, fragment tolerance 0.8 Da, missed cleavages: 3, instrument type: ESI-TRAP. The interpretation and presentation of MS/MS data was performed according to published guidelines. 49 Protein ID is also based on the assignment of at least two peptides. In the case where proteins were identified based on one peptide sequence, the corresponding MS/MS spectra were inspected and verified manually.

3 Cell Line Exosome Production (μg exosomes/mg total cell protein) U87 control U87 Dll HUVEC control HUVEC treated with Dll Table S2. Level of exosome production Exosomes were isolated from U87, U87-Dll4, HUVEC and HUVEC seeded on Dll4- coated plates. The exosomes were then lysed in RIPA. The exosome producing cells were also lysed and the protein content of the two lysates assessed. Exosome production is expressed as μg of exosomes per mg of total cell protein.

4 Protein Name Accession Number (Swissprot) Score (PLGS) 1 Seq. Cov. Nr. of MS/MS (%) 2 queries 2 EPHB2 receptor P PDGF receptor P Insulin Receptor P BMP3b P BMP4 P DLG1 Q Score based on Mascot assignment, and is a statistical measure of accuracy of assignation, a higher score implies greater confidence of protein identity 2 Sequence coverage (in %) and number of MS/MS queries are taken from one of the three triplicate analytical runs. Table S3. Quantitative, comparative mass spectroscopy 100μg of U87 exosomes were extracted by chloroform/methanol precipitation and subjected to quantitative, comparative mass spectroscopy analysis as described in the methods. Angiogenic proteins only identified in the Dll4-containing exosomes are listed along with a previously identified Dll4 interacting protein. For MS E data, MS/MS spectra were reconstructed by combining all masses with identical retention times. The UniProtKB/Swiss-Prot database was searched for each run with the following parameters: peptide tolerance, 100 ppm; fragment tolerance, 0.1 Da; trypsin missed cleavages, 2; fixed modification carbamidomethylation, variable modification: Met oxidation. Alternatively MS/MS spectra (peak lists) were also searched against the above database using Mascot version (Matrix Science) and the following parameters: peptide tolerance, 0.2 Da; 13 C = 2; fragment tolerance, 0.1 Da; missed cleavages, 2; instrument type, ESI-Q-TOF. All database searches were restricted to human species because of the complexity of the searches when combined with multiple modifications. The interpretation and presentation of MS/MS data were performed according to published guidelines. 49 The analysis of quantitative changes in protein abundance, which is based on measuring peptide ion peak intensities observed in low collision energy mode in a triplicate set, was carried out using Waters Expression Analysis Software (WEPS TM ), which is part of PLGS (Expression version 2) as described previously. 17

5 Figure S1. Exosomes increase HUVEC proliferation HUVEC were seeded in triplicate in 96-well plates at a density of 3000cells/well. After a 16 hour incubation in MI99+2% FCS starvation media, 50μg/ml of control or Dll4 exosomes were added and the proliferation assessed at 48 and 72 hours post addition using the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay from Promega according to manufacturers instructions. Data was analyzed by t-test using GraphPad Prism 4.0b software and shown as mean with SD.

6 Figure S2. Effect of exosomes on tumour size and vessel density U87 xenografts were grown and injected three times a week for five weeks with PBS, control exosomes or Dll4 exosomes (isolated from U87 cells) at a concentration of 50μg/ml. The animals were then sacrificed and the tumours measured and sectioned for vessel staining. A Chalkley vessel count (CVC) was performed for vessel density. Oneway ANOVA with a Tukey s Multiple Comparison post-test were performed using GraphPad Prism 4.0b software.

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8 Figure S3. The hdll4 antibody does not detect mdll4 (A) U87 xenografts overexpressing mdll4 were stained with anti-human Dll4 to confirm specificity. (B) Western blot confirming the expression of mdll4 in the U87 cell line. (C) Higher magnification images of U87 hdll4 xenografts showing location of hdll4 and transfer of protein to red blood cells. (D) Higher magnification of U87 xenografts not expressing Dll4 stained with anti-human Dll4 antibody to confirm that the antibody does not non-specifically bind to red blood cells. Red arrows indicate RBC. Images acquired using a Zeiss Axioskop 2 microscope at 100x magnification.