Hsp70

Cells and exosomes were lysed in lysis buffer (Beyotime, #P0013, China) supplemented with protease inhibitor cocktail (Roche, USA) and PMSF (sigma, USA). Equal amounts of protein were loaded to SDS-PAGE gel, then transferred to PVDF membrane with 0.22 um pore size (Milipore, USA). The PVDF membrane was blocked with skimmed milk, incubated with primary antibody in 4°C overnight, then washed three times with TBS-T. Each PVDF membrane was incubated with secondary antibody in room temperature for 1 hour and added ECL western HRP substrate (Milipore, USA). The following primary antibodies were used: CD9 (SBI, #EXOAB-CD9A-1, 1:1000, USA), CD63 (SBI, #EXOAB-CD63A-1, 1:1000, USA), HSP70 (SBI, #EXOAB-HSP70A-1, 1:1000, USA), TSG101 (Proteintech, #14497-1-AP, 1:500, USA), Alix (Proteintech, # 12422-1-AP, 1:500, USA), HIF-1α (CST, # 36169S, 1:1000, USA), β-Actin (Proteintech, #66009-1-Ig, 1:5000, USA), GAPDH (Proteintech, #60004-1-Ig, 1:5000, USA), GJA1 (CST, #83649S, 1:1000, USA).

EVs were separated and concentrated from 500 µl of plasma as described above and the pellet was directly lysed in boiling Laemmli for 10 min. Proteins were resolved by SDS-PAGE, transferred onto nitrocellulose membranes and blotted with the following antibodies, all diluted at 1/1000: CD9 (EXOAB-CD9A-1, SBI), CD63 (EXOAB-CD63A-1, SBI), VWF (sc-53466, Santa Cruz), FCN3 (CL7767AP, Cedarlane), Apolipoprotein A1 (sc-376818, Santa Cruz), HSP70 (EXOAB-HSP70A, SBI), and GM130 (ab52649, Abcam) diluted at 1/1000. Membranes were incubated with HRP-conjugated secondary antibodies (Southern Biotech), diluted at 1/5000 and then revealed by chemiluminescence. Acquisitions were performed with Fusion software, version FX7 16.15 (Vilbert Lourmat, Collegien, France). Uncropped blots are available in Fig. S1.

Tumor cells were cultured in RPMI 1640 media supplemented with 10% exosome-depleted FBS (BI, Israel). Supernatant of tumor cells culture was collected 48 hours after cell reached 80% confluence. Then the supernatant was centrifuged at 4000 rpm for 2 hours to remove cell debris, followed by 4000 rpm centrifuge for 30 min using 100 KDa MWCO to make the exosome-concentrated solution. The exosome was isolated by exosome quick extraction solution. The protein content of exosome was determined by BCA protein assay kit. The characterization of exosomes was confirmed by measuring expression of exosome-specific markers ALIX, HSP70, TSG101, CD81 and CD9 (SBI) by western blot analysis and Transmission Electron Microscopy (FEI spirit 120kV, USA). Exosomes were labeled with PKH67 membrane dye at 37°C for 5 minutes, and analyzed by confocal microscope (FV1000, Olympus). All the unspecified exosomes used in this study were from either TC-1 or MC38 tumor cells.

Western blotting was performed as previously described [6 (link)]. Briefly, total protein concentration in the cell lysate was determined by using DC™ protein assay (Bio-Rad Laboratories, Hercules, CA). Fifty micrograms of total protein were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred to a nitrocellulose membrane. The membrane was incubated with blocking buffer (Thermo Fisher) for 1 h at room temperature and incubated overnight at 4°C with the primary antibody to SNAIL1, E-cadherin (1:1000 dilution, Cell Signaling), IGF1R (1:1000 dilution, Abcam, Cambridge, MA, USA), CD9, CD63 or HSP70 (1:1000 dilution, SBI System Biosciences, Mountain View, CA). The membrane was washed with Tris-buffered saline-Tween-20 buffer (TBST) and incubated with HRP-conjugated secondary antibodies (1:10,000 dilution, Abcam) at room temperature for 1h. After washing with TBST immunoreactivity was detected using enhanced chemiluminescence reagent (GE Healthcare Bio-Sciences, Marlborough, MA). β -actin, the loading control, was detected using β -actin antibody (1:10000, Abcam) and anti-mouse IGG (1:10000, Sigma Aldrich) as the primary and secondary antibodies, respectively.

Isolated Exos were confirmed via flow cytometry analysis with CellTrace™ Violet (ThermoFisher Scientific, Waltham, MA, USA) as previously published [26 (link)]. In brief, isolated Exos were incubated with CellTrace reagent for 20 min at 37 °C. Following labeling, flow cytometry analysis was accomplished using the CytExpert Software v2.5 for the CytoFLEX Flow Cytometer (Beckman Coulter, Brea, CA, USA).
Exo pellet was then characterized through a western blot for specific protein biomarkers for exosomes, namely CD63 and heat shock protein 70 (HSP70), as published [9 (link)]. In brief, exosomal proteins were extracted by reconstitution with radioimmunoprecipitation assay lysis buffer (RIPA), and the concentration of isolated protein was acquired using the Bio-Rad protein assay. The extracted protein (50 μg) and ladder (SeeBlue Plus2 Prestained Standard) [38 (link)] were loaded onto 4–12% Bolt gels and ran for 22 min at 200 V, followed by transfer to a polyvinylidene difluoride (PVDF) membrane using the iBlot2 Gel Transfer Device. Membranes were then incubated with antibodies CD63 (1:1000; SBI, Palo Alto, CA, USA) and HSP70 (1:1000; SBI, Palo Alto, CA, USA).