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BDX2, a BDX Rat Fibrosarcoma Cell Line (1) Was Maintained in DMEM/10% 1 Supplementary Material and Methods Additional cell lines: BDX2, a BDX rat fibrosarcoma cell line (1) was maintained in DMEM/10% FCS, MatLyLu and AT6.1, rat prostate carcinoma lines (2) were maintained in RPMI1640/10% FCS supplemented with 250nm dexamethasone. AT6.1 cells have been stably transfected with Tspan8 cDNA (AT6.1-Tspan8). LuFb (BDX lung fibroblast line) and RAEC (rat aortic endothelial cell line, Cell-Lining, Berlin, Germany) were maintained in RPMI1640/10% FCS. 2 Supplementary Figure Legends Suppl.Figure 1 Electron microscopy of AS- and AS-Tspan8-exosomes: Cells grown in plastic dishes were fixed with 2% glutaraldehyde and ferrocyanide-reduced osmium, contrasted en bloc with uranyl acetate, dehydrated with ethanol and embedded in resin. Ultrathin sections were contrasted and viewed with an EM 10 CR electron microscope (Carl Zeiss NTS, Oberkochen, Germany). Exosome-enriched fractions were negatively stained with 1% uranyl acetate. Electron microscopy of AS-Tspan8 cells (top), a crude exosome preparation (lower left) and the pooled d1.141-d1.171 sucrose gradient fractions (lower right). The crude preparation contains besides cup-shaped exosomes non-exosome vesicles, which are not seen in the sucrose gradient fraction. Scale bar: top: 250nm, bottom: 50nm. Suppl.Figure 2 Tetraspanin and integrin expression in rat tumor cell lines: (A) AS and two AS-Tspan8 clones were tested for Tspan8 expression by flow cytometry. Overlays of the negative control (black line) and D6.1 staining (gray line) are shown). (B) Expression of Tspan8, CD49c and CD49d was evaluated by flow cytometry in AS, AS-Tspan8, AT6.1, AT6.1-Tspan8, BDX2 and MatLyLu cells. The mean percentage of stained cells (left) and the mean intensity of staining (right) are shown. For the mean intensity of staining the values of the negative controls have been subtracted. Suppl.Figure 3 Tspan8-exosomes promote EC migration: (A) Subconfluent monolayers of AoR-EC and LuFb were scratched and medium was exchanged containing no exosomes or 10µg/ml of AT6.1-, AT6.1-Tspan8-, BDX2- or MatLyLu-exosomes. Wound healing was evaluated after 24h, 48h and 72h. Black dotted line: scratch; white dotted line: healing; (scale bar: 500µm). RAEC migration was only accelerated in the presence of exosomes derived from Tspan8-expressing cells (AT6.1-Tspan8 and BDX2). LuFb migration was independent of Tspan8. It was promoted by MatLyLu-exosomes, which do not express Tspan8 and CD49d. (B) AoR-EC were plated on Matrigel and cultured in the presence of medium, VEGF, AS-, AS-Tspan8-, AT6.1-, AT6.1-Tspan8-, BDX2- or MatLyLu-exosomes. Branching was evaluated after 24h. Microscopic appearance (scale bar: 1mm) and the mean±SD (10 fields) branching nods are shown. VEGF, AS-Tspan8-, AT6.1-Tspan8-, and BDX2-exosomes strongly support branching. AS-exosomes do not, AT6.1- and MatLyLu-exosomes poorly support branching. Significant differences as compared to the medium control and between Tspan8+ vs. Tspan8- exosomes are shown. Suppl.Figure 4 Schematic presentation of marker profile during EC progenitor maturation: A modified summary according to several reviews (58-61) is shown. 3 Supplementary Table 1 List of Antibodies Antibody Supplier Antibody Supplier CD9 BD, Heidelberg, G.* CD106 Biozol, Eching, G. CD11b clone Ox42 (EAACC)† CD133 Santa Cruz, Heidelberg, G. CD31 BD, Heidelberg, G. CD151 (3)‡ CD49a BD, Heidelberg, G. CD184 Santa Cruz, Heidelberg, G. CD49b BD, Heidelberg, G. Bag6 § CD49c BD, Heidelberg, G. HSP70 Abcam, Cambridge, UK CD49d BD, Heidelberg, G. Lamp1 BD, Heidelberg, G. CD49e BD, Heidelberg, G. MAC-2BP Santa Cruz, Heidelberg, G. CD61 Biozol, Eching, G. Tspan8 clone D6.1 (4)‡ CD71 clone Ox26(EAACC)† VEGFR1 Biotrend, Cologne, G. CD81 Santa Cruz, Heidelberg, G. VEGFR2 Biotrend, Cologne, G. * G.: Germany † EAACC: European Association of Animal Cell Cultures, Porton Down, UK ‡ References in Suppl. Table 6. § Kindly provided by C. Cziepluch, DKFZ, Heidelberg, Germany 4 Supplementary Table 2 List of Primers A. RT-PCR gene 5’-sequence-3’ CCR1 fw. GGC AGG GAT TCA CTT CAA GA CCR1 rev. GCC ATC GGT GCA ATC TAT CT CD31 fw. TGA GTA CCA GCT GAC GGT GAA C CD31 rev. TGT CCA CTG TGC TCT ACC AAG G CD131 fw. ACC CAA GAC ACC TTC AAT GC CD131 rev. ACG GTG TTG AGT TCC CTG TC CXCL1 fw. GGC AGG GAT TCA CTT CAA GA CXCL1 rev. GCC ATC GGT GCA ATC TAT CT CXCL5 fw. GTT CAC ACT GCC ACA GCA TC CXCL5 rev. TTA TCA ACG GAG CTT CTG GG DEC1 fw. AAC TTA CAA ATT GCC GCA CC DEC1 rev. ACT GGC ACA CAG TTT TTC CC FGFbp 1 fw. ACG GAA TAA GCA GAG GAG CA FGFbp 1 rev. TTC CTC TGG GTG AGG ACA TC GAPDH fw GAC CCC TTC ATT GAC CTC AAC GAPDH rev. CTT CTC CAT GGT GGT GAA GA GDF3 fw. GTT CAG GAT CGT GGT GTG TG GDF3 rev. ACA GCT TGG TGG GGA TAC AG HMOX1 fw. AGC ATG TCC CAG GAT TTG TC HMOX1 rev. AGG TAG CGG GTA TAT GCG TG MIF fw. CAG TGC ACG TGG TCC CGG AC MIF rev. GTA ATA GTT GAT GTA GAC CCG MycN fw. ATC ACT GTA CGC CCC AAG AC MycN rev. CCT TTG GTG GAA CGA CAC TT SMA fw. ACT GGG ACG ACA TGG AAA AG SMA rev. GTC CAG AGC GAC ATA GCA CA Spi-C fw. CAC AGA CTT TCG TCC AGC AA Spi-C rev. GAC GGA GCA AGT CTT TGG AG SRF fw. ACG ACC TTC AGC AAG AGG AA SRF rev. GGC TTC AGT GTG TCC TTG GT 28S rRNA fw. GAA TTC ACC AAG CGT TGG ATT G 28S rRNA rev. TGG GCG GGA TTC TGA CTT AGA TCEB1 fw. TGG ATG GAG AGG AGA AAA CC TCEB1 rev. CTA AGG AGT TCA CGG CCA TC TF fw. ACA AAT GCA CTG GAA CCA CA TF rev GTC AGC CTC CTC GTC TAT GC Tspan8 fw. CAG GTA CCG CCA CCA TGG CAG GTG T Tspan8 rev. GTC TCG AGT CAT TTG CTT CCA ATT TGG VE-Cadherin fw. GTG CTC CAA GGA GAA CAA GC VE-Cadherin rev. CTG TGA TGT TGG CGG TAT TG VEGFa fw. GCC CAT GAA GTG GTG AAG TT 5 VEGFa rev. AAT GCT TTC TCC GCT CTG AA VEGFc fw. CTA CAG ATG TGG GG TTG CT VEGFc rev. CAG GCA CAT TTT CCA GGA T VEGFR1 fw. GGC CTG TGG AAG GAA TAA CA VEGFR1 rev. TGT ATT GAG GTC CGT GGT GA VEGFR2 fw. ACA GTT CCC AGA GTG GTT GG VEGFR2 rev. TCT CCG GCA GAT AGC TCA AT vWF fw. GGC AGG ACG TTC TAT GGT GT vWF rev. CAC CAC AAA AGC TTT GAG CA B. Q-PCR gene 5’-sequence-3’ Q-CD31 fw. CGA AAT CTA GGC CTC AGC ACT Q-CD31 rev. CTT TTT GTC CAC GGT CAC CT Q-CD133 fw. TGC TCA TGA GTC TTG GCA TC Q-CD133 rev. TGT GTT GTA TTG CCC CAG AA Q-CD146 fw. CAG CAA AGG AGA GGA AGG TG Q-CD146 rev. GAG TCA GGT GTG AGG GTG GT Q-CD184 fw. CCA GGG CTG GAG AGC GAG CA Q-CD184 rev. GGT CAG CCA CGG ACA GGT GC Q-VEGFR2 fw. ACA GTT CCC AGA GTG GTT GG Q-VEGFR2 rev. GTC ACT GAC AGA GGC GAT GA Q-vWF fw. CTG GCC AGG GAC CCT CTG CAC Q-vWF rev. TGG CAG TCC CCA GCC AGA AGA 6 Supplementary Table 3 A Differential mRNA expression in AS- vs. AS-Tspan8-exosomes AS- AS- Tspan8- Tspan8- Genes (accession No)* vs. AS- Genes (accession No) vs. AS- exosomes† exosomes† LOC499080 putative nuclein acid Dnmt3b (NM_001003959) 6.4 binding proteins (XM_574364) 46.9 LOC502207 (XM-577667) 6.4 LOC500207 similar to ribosomal LOC367402 (XR_007623) 6.4 proteins (XM_575559) 13.5 LOC689811 (XM_001072103) 6.3 LOC500211 putative nucleic acid LOC502465 (XM_577945) 6.3 binding protein (XM_575563) 13.0 RGD1566233-pred. (XM_575213) 6.3 LOC498905 putative nucleic acid LOC501517 (XM_576918) 6.2 binding protein (XM_574194) 11.7 RGD1560140-pred. (XM_577949) 6.1 LOC502728 XMAP4 -micro- LOC500663 (XM_576041) 6.0 tubule-assoc. protein 4 Sod3 (NM_012880) 5.9 (XM_578227) 11.3 RGD1306073-pred. (XM_343599) 5.9 LOC497864 no similarity Tm4sf2-mapped (XM_343768) 5.9 (XM_579753) 10.2 LOC499503 (XM_574827) 5.9 RGD1565815-pred. (Elongin A) MGC116197 (NM_001025755) 5.9 (XM_573888) 9.9 LOC684696 (XM_001071647) 5.8 RGD1560826-pred. GAPDH RGD1561059-pred. (XM_227659) 9.9 (XM_001060093) 5.8 LOC361157 putative RNA binding RGD1562317-pred protein 1 (XM_341443) 9.7 (XM_001063359) 5.7 LOC502280, similar to LOC500311 (XM_575661) 5.7 MAPK2/PAR-1 (XM_577741) 9.2 LOC501634 (XM_577031) 5.6 LOC502517 similar to Trypanos. LOC367582 (XM_578908) 5.6 brucei TREU927 hypothetical Fgfbp1 (NM_022603) 5.6 protein (XM_578001) 8.6 Cpne9 (NM_001024982) 5.5 RGD1561305 (XM_580123) 8.4 LOC498567 (XM_573842) 5.5 RGD1559440-pred. CAPS2-se- LOC499054 (XM_579916) 5.5 cretion activator 2 (XM_231528) 8.2 Spetex-2D (NM_001011701) 5.4 Cndp2 – cytosolic nonspecific RGD1563709-pred. (XR_007499) 5.4 dipeptidase 2 (NM_001010920) 8.1 LOC498765 (XM_574051) 5.4 LOC500427 (XM_575789) 7.5 RGD1308723-pred. LOC302573 unknown protein (XM_001079278) 5.3 (XM_228795) 7.4 RGD1562893-pred. (XM_577935) 5.2 LOC499039 similar to kinesin Smptb (NM_182818) 5.2 family member 25 (XM_574324) 7.2 LOC500969 (XM_576377) 5.2 RGD1561656-pred. similar to LOC498467 (XM_573724) 5.1 putative mitochondrial protein RGD1565591-pred. (2B324) (XM_344647) 7.1 (XM_001077382) 5.1 LOC316848 similar to SPIN-2 Sema5a-pred. (XM_241275) 5.0 protein (XM_229302) 6.8 Slc35f5-pred. (XM_222576) 5.0 RGD1304762 (NM_001034918) 6.8 Spetex-2C (XM_573726) 5.0 RGD1562211-pred. LOC498935 (XM_574221) 5.0 (XM_001066361) 6.8 LOC306038 (XM_224366) 5.0 RGD1562268-pred. LOC498568 (XM_573843) 4.9 (XM_001072983) 6.6 LOC311772 (XM_231028) 4.9 LOC688644 (XM_001067734) 6.6 Nagk-predicted (XM_216188) 4.9 Olr1704-pred.
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