Protein Name Accession Number (Swissprot) Sequence Coverage (%) No. of MS/MS Queries Mascot Score 1 Reference Cytoskeletal proteins Beta-actin P60709 37 14 298 Alpha-actin P68032 33 10 141 20 Beta-actin-like protein 2 Q562R1 11 4 168 43 Profilin-1 P07737 21 2 51 UD 2 Adhesion Integrin-beta-1 P05556 18 13 294 UD Integrin-alpha-3 P26006 7 7 158 UD Integrin-alpha-5 P08648 1 1 75 UD CD44 P16070 5 4 116 44 Galectin-1 P09382 17 2 82 45 Enzymes Glyceraldehyde-3-phosphate P04406 41 10 280 UD dehydrogenase Alpha-enolase P06733 14 5 174 20 Beta-enolase P13929 7 2 157 Fructose-bisphosphate P04075 3 1 80 UD aldolase A Pyruvate kinase P14168 4 1 55 UD L-lactate dehydrogenase B P07195 4 2 65 UD chain 5 -nucleotidase P21589 24 11 296 UD Sodium/potassiumtransporting P05023 9 10 195 43 ATPase subunit alpha-1 Sodium/potassiumtransporting ATPase subunit beta-3 P54709 8 2 88 UD Signalng proteins G(i) alpha-3 P08754 18 5 120 UD G(i) alpha-2 P04899 13 4 98 UD G-protein beta-1 P62873 10 3 46 UD G-protein beta-4 Q9HAV0 12 2 30 G-protein beta-2 P62879 10 3 57 43 14-3-3 P63104 17 3 152 20 Prostaglandin F2 receptor negative regulator Q9P2B2 1 1 75 46 Membrane transport and fusion Rab-15 P59190 2 1 65 UD Rab-35 Q15286 5 1 65 UD Annexin A2 P07356 14 4 110 UD Myristoylated alanine-rich protein kinase C substrate P29966 9 2 50 UD 20
Protein Name Accession Number (Swissprot) Sequence Coverage (%) No. of MS/MS Queries Mascot Score 1 (MARCKS) ADP-ribosylation factor 4 P18085 6 1 71 Reference 47 Tetraspannins CD63 P08962 4 1 54 UD CD151 P48509 3 1 53 48 Others Collagen alpha-3 P12111 1 4 176 UD Collagen alpha-1 P12109 1 1 73 UD Collagen alpha-2 P12110 1 2 64 47 Solute carrier family 16, P53985 2 1 44 43 member 1 Basigin/CD147 P35613 7 2 83 30 Elongation factor 1-alpha P68104 2 1 68 UD Brain abundant, membrane P80723 10 2 48 UD attached signal protein 1 Solute carrier family 29 Q99808 5 1 46 1 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 (http://dir.nhlbi.nih.gov/papers/lkem/exosome/) 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.
Cell Line Exosome Production (μg exosomes/mg total cell protein) U87 control 142.2 U87 Dll4 95.67 HUVEC control 17.68 HUVEC treated with Dll4 6.16 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.
Protein Name Accession Number (Swissprot) Score (PLGS) 1 Seq. Cov. Nr. of MS/MS (%) 2 queries 2 EPHB2 receptor P29323 35.89 9.0 7 PDGF receptor P09619 84.2 15.5 12 Insulin Receptor P06213 49.33 11.0 9 BMP3b P55107 45.73 13.6 6 BMP4 P12644 27.67 12.0 3 DLG1 Q12959 37.96 11.0 8 1 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 2.2.04 (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 2.2.5 (Expression version 2) as described previously. 17
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.
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.
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.