Ab133586

Fibroblasts were derived from human foreskin explants and cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, USA, 11885092), The concentration of glucose in the culture medium was 5.5 mmol/L in normal glucose (NG) group, and 30 mmol/L in high glucose (HG) group. After 5 days, the cultured medium was collected every 48 h from passage 3 to passage 7. The supernatants of these cells were mixed together. EVs were collected from supernatant by a series of ultra-high-speed centrifugation procedures according to the steps reported in literatures (13 (link)).
The morphology of NG-EVs and HG-EVs was observed by transmission electron microscope (TEM) (Hitachi, Japan) and the particle size was measured by nanoparticle tracking analyzer (Particle Metrix GmbH, Germany). Expressions of EV positive markers CD63 (abcam, ab68418), TSG101 (abcam, ab133586), and negtive marker calnexin (abcam, ab133615) were analyzed by Western blot. A brief step of uptake assay is as follows. The membrane of EVs was labeled with PKH67 (green). HUVECs were counterstained by actin (cytoskeleton, red) and DAPI (cell nucleus, blue). The internalization process was observed under confocal microscope (Leica, Germany).

Lysis buffer was determined by radioimmunoprecipitation and protein concentration by the bovine serum albumin method. Proteins were isolated from the lysate by SDS-PAGE and transferred to a PVDF membrane. After containment, specific primary resistance to PKM2 (1/1000; ab85555; Abcam, USA), HK2 (1/1000; Ab133691), LDHA (1/5000; Ab52488), CD36 (1/1000; Ab133625), TSG101 (1/1000; Ab133586), PARP (1/1000; Ab32071), Bax (1/1000; Ab32503), bcl-2 (1/2000; Ab182858) or beta-actin (1/200; Ab115777) for incubation followed by peroxidase-coupled secondary resistance for incubation. Final detection of protein traces using an improved chemiluminescence detection system (Pierce, USA).

Exosome isolation was carried out following a previously published protocol [25 (link)]. Briefly, hucMSCs were cultured and reached 50–60% confluence. They were then placed in a serum-free MSC medium (Nuwacell Biotechnology, RP02010-1 and RP02010-2, China). After 48 h of cultivation, the medium was obtained and centrifuged for 10 min at 300g and for 30 min at 10,000g. Next, the supernatant was filtered with a 0.22-μm filter (Millipore Corp, USA) and further ultracentrifuged at 100,000 g for 70 min in an SW32Ti rotor (Beckman Coulter, USA). After removing the supernatant, the pellet was resuspended in PBS and then centrifuged at 100,000g for 70 min again. Finally, the pellet was stored at − 80 °C after being collected in 200–300 μL of PBS.
The particle size distribution of hucMSC-Exo was analyzed using a nanoparticle tracking analysis system (ZetaView, Germany). The characteristic morphological observation of the exosomes was performed with a transmission electron microscope (FEI Tecnai Spirit G2). Western blot analysis was performed to identify the expression of exosome-specific markers TSG101 (ab133586, Abcam, 1:1000 dilution), CD9 (A1703, Abclone, 1:1000 dilution), CD63 (25682-1-AP, Proteintech, 1:1000 dilution), and heat shock protein HSP70 (A12948, Abclone, 1:1000 dilution).

To detect the presence of tetraspanin proteins (TSG101 and CD81) in membrane, lipogenesis-related proteins (PPARγ and CEBPα) and angiogenic-related proteins (Ang-1 and VEGF) in the contents, proteins were harvested from SVF-EVs contents with RIPA Lysis Buffer (Beyotime, Hainan, Jiangsu, China) supplemented with phenylmethyl sulfonyl fluoride (PMSF) protease inhibitor. Denatured protein samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membranes. Then membranes were blocked with 5% skimmed milk, and incubated with specific antibodies against TSG101 (ab133586, Abcam, UK), CD81(ab109201, Abcam, UK), PPARγ (sc-7273, Santa, USA), CEBPα (sc-365318, Santa, USA), Ang-1 (23302-1-AP, Sanying, China) and VEGF (66828-1-Ig, Sanying, China) overnight at 4 °C. The membrane was then incubated with secondary antibodies which are purchased from GeneTex (Irvine, USA). Protein bands were visualized by the FluorChem Imaging System (ProteinSimple, San Jose, USA) using the Amersham Hyperfilm ECL reagent.

The whole cell lysates were harvested via cell lysis buffer and the protein concentration was detected with BCA Protein Assay Kit (Thermo). Then the proteins were undergoing separating by 8–12% gradient gels and transferred to PVDF (Polyvinylidene Fluoride) membranes. Membranes were blocked by blocking buffer and incubated with primary antibodies at 4 °C overnight. At last the membranes were incubated with secondary antibodies at room temperature for 1 h and scanned by infrared imaging system. The following primary antibodies were used: TSG101 (1:1000, ab133586, Abcam), CD81 (1:1000, ab109210, Abcam), HUR (1:1000, ab200342, Abcam), HER-2 (1:1000, ab134182, Abcam), GAPDH (1:1000, 60,004–1-Ig, Proteintech), p62 (1:1000, cat. no. 18420–1-AP), LC3 (1:1000, cat. no. 14600–1-AP), CD63 (1:1000, BD Bioscience, clone H5C6), CD9 (1:1000, Millipore, clone MM2/57), Fibronection (1:1200, ab285285, Abcam).

Cell proteins were extracted with RIPA lysis buffer (Thermo Fisher, USA). The protein was extracted by incubation, vortex, and centrifugation (15,000 ×g, 4°C for 25 min). A BCA reagent kit (Beyotime, China) was used to measure the protein concentrations. Total protein was separated by SDS-PAGE gel (100 V, 1.5 h) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA) (50 V, 80 min). After blocking in 5% nonfat milk for 1 hour, the membranes were incubated overnight at 4°C with the indicated primary antibodies, including anti-CD63 (Abcam, ab68418), anti-TSG101 (Abcam, ab133586), anti-E-cadherin (Abcam, ab76055), anti-N-cadherin (Abcam, ab76011), anti-vimentin (Abcam, ab20346), anti-JAK1 (Abcam, ab138005), anti-STAT3 (Abcam, ab68153), IL-10 (Abcam, ab215975), arginase-1 (ab133543) and anti-GAPDH (Abcam, ab9485). They were then incubated with secondary antibodies for 1 hour at room temperature and visualized by the ECL chemiluminescence reagent (Millipore, USA).

HUVECs were lysed in cold 1 × Cell Lysis Buffer (Cell Signaling Technology, USA) supplemented with 1 mM PMSF (Sigma) at 4 °C for 30 min. The M-EVs and N-EVs were lysed in 20 μL lysis buffer at 4 °C for 10 min. Total protein concentration was determined using the BCA protein assay kit (Pierce, USA). Western blotting was performed according to the standard protocol, as our previously described [38 (link)]. The antibodies used were as follows: CD63 (1:1000, ab134045, Abcam), TSG101 (1:1000, ab133586, Abcam), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1000, 5174, Cell Signaling Technology), EFNA (1:1000, 33,810, Signalway Antibody). The secondary antibody was horseradish peroxidase-conjugated antibody. Protein bands were visualized by chemiluminescence using enhanced chemiluminescence substrate (Millipore Corporation, Bedford, MA) and the blots were analyzed using Image J software (NIH, USA).