Exoab kit 1

Purification of exosomes was confirmed by PS Capture Exosome ELISA Kit (Wako) according to the manufacturer’s instructions. Antibodies used as capture reagents were as follows: anti-CD63 contained in the above kit, and anti-CD81, anti-CD9 and anti-Hsp70 contained in the EXOAB-KIT-1 (System Biosciences). For detection of the latter three antigens, we used a goat anti-rabbit IgG HRP-conjugated secondary antibody.

Western blotting for the EV markers CD63, CD9, and HSP70 (EXOAB-KIT-1, System Biosciences), TSG101 (ab83, Abcam), and ALIX (ab117600, Abcam) was undertaken as previously described by Lötvall et al. (63 (link)). The purity of EV samples was tested for contaminating intracellular organelles by ATP5A (mitochondria) (15H4C4, Abcam), nuclear fractions by histones (histone H3) (D1H2/4499P, Cell signaling), and platelets by CD41 (ab63983, Abcam). Briefly, protein quantity in EVs was detected by lysing EV pellets in CelLytic MT Cell LysisReagent or RIPPA buffer (Cell Signaling) with added protease inhibitors (Roche Complete). To ensure equal loading, protein concentration was measured by bicinchoninic acid assay (Thermo Scientific), and 10–40 μg protein was loaded per well. SDS page samples were separated on 4%–12% bis-tris gradient gels (Life Technologies) under nonreducing conditions. Separated samples were transferred to PVDF or nitrocellulose membranes, and nonspecific binding was blocked with 5% milk in PBS-Tween. Membranes were incubated with antibodies overnight, and membranes were washed and incubated with a secondary antibody conjugated to horseradish peroxidase. Membranes were washed again before incubation with enhanced chemiluminescence substrate (Pierce ECL, ThermoFisher Scientific) and imaged on a Bio-Rad ChemiDoc MP Imaging system.

MDA-MB-231 cells were purchased from the cell bank of the Chinese academy of sciences (Shanghai, China). No contamination of mycoplasma was confirmed by PCR test. Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin and streptomycin at 37 °C in a humidified atmosphere containing 5% CO₂.
DMEM, FBS, the enhanced chemiluminescence (ECL) reagent, and Total exosome isolation reagents were purchased from Thermo Fisher Scientific (4478359; Waltham, MA, USA). Matrigel was purchased from BD Biosciences (San Josè, CA, USA). PKH26 Red fluorescent cell linker kit was purchased from Sigma Chemicals (St. Louis, MO, USA). TRIzol reagent was obtained from Invitrogen (Carlsbad, CA). The antibodies specific for phospho-c-Raf (#9427), phospho-ERK (#4377), phospho-p38 (#9211), phospho-Akt (#9271), Akt (#9272), phospho-mTOR (#2971), STIM1 (#A5668) β-actin (#4967), and the horseradish peroxidase-conjugated goat anti-rabbit secondary antibody were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The anti-IRS1 (ab40777) and anti-CD31 (ab28364) antibodies were obtained from Abcam (Cambridge, MA, USA). The exosome-specific primary antibodies including CD9, CD63, CD81, and HSP70 were obtained from System Biosciences (EXOAB-KIT-1, Palo Alto, CA, USA).

Measurement and analysis of exosomes by Nanosight using approximately 1000 μl appropriately diluted in PBS (1000 times) exosomes preparations was performed on a NanoSight ns300 Malvern (Worcester, UK). Individual videos of 60 s for each sample were acquired using the maximum camera gain and analyzed by the NanoSight particle tracking software to determine particles density and size. Exosomes were fixed in 2% paraformaldehyde at 4 °C and analyzed using Electron microscope. Exosomes were then deposited onto formvar grid for 20 min. The grids were then fixed (1% glutaraldehyde for 5 min), washed with distilled water and negatively stained with 4% uranyl acetate and 2% methyl cellulose for 5 min. The grids were then thoroughly dried and subsequently observed by transmission electron microscope (Tecnai G2 spirit). The diameters of the exosomes were quantified from the obtained micrographs (Supplementary Figure S5). Analysis for the presence of exosomes special markers CD63, CD9, CD81 and Hsp70 antibodies (System Biosciences catalog no. EXOAB-KIT-1) was carried out using western blot.

HDFs or EVs were lysed in Laemlli buffer. The protein in the lysate was quantified using the BCA protein assay, electrophoresed in SDS-PAGE gel and transferred to PVDF membrane. The membrane was incubated with primary antibodies to a-SMA (ab32575, Abcam), collagen type I (ab34710, Abcam), Bcl2 (sc-509, Santa Cruz Biotechnology), MMP2 (ab97779, Abcam), vinculin (V4139, Sigma,), GAPDH (sc-32233, Santa Cruz Biotechnology), b-actin (4970s, Cell Signaling Technology, Leiden, The Netherlands), or exosome markers (HSP70, CD81, CD63, CD9; EXOAB-KIT-1, System Biosciences, Palo Alto, CA, USA) overnight at 4 °C. After washing in TBST, the blots were incubated with horseradish peroxidase (HRP)-labeled secondary antibodies (P-RAM Iss, P-RAQ Iss, Imtek) for 1 h. The labeled proteins were visualized with ChemiDocTM Touch imaging system (Bio-Rad Laboratories, Hercules, California, USA) using an enhanced chemiluminescence (ECL) kit (Pierce, Waltham, MA, USA).