Exo fect exosome transfection kit

Purified ELV derived from control HDFn were transfected with ND1, ND5, SLCO2B1, and SEPIN3 using Exo-Fect Exosome Transfection Kit (System Biosciences) according as per the manufacturer’s instructions. Following the same protocol, purified exosomes derived from control mouse serum were transfected with mND1, mND5, mSLCO2B1, and mSEPIN3. Briefly, PCR products were run on 1.5% agarose gel, cut at the desired length, and purified using Wizard SV Gel and PCR Clean-Up System (Promega). After purification of DNA, 50 μl of purified ELV PBS suspension were mixed with 10 μl of Exo-Fect solution and 20 μl of 0.2 μg of each amplified DNA. Transfected ELV were added to cells plated in 2-well chamber slides (1 × 10⁵ cells/well) and incubated for 24 h. Further, to observe the uptake of ELV, ELV containing amplified DNA were labelled with PKH-67 using the PKH-67 labeling kit according to manufacturer’s recommendations (Sigma-Aldrich).

Inhibitor and mimics of miR-25-3p were purchased from GenePharma. The sequences of inhibitor and miRNA mimics referred above were listed in Supplementary Table 1 and Supplementary Table 2. Lentivirus vectors expressing miR-25-3p and repressing miR-25-3p were constructed and generated by Genechem Inc. In the rescue experiments, cells that stably expressed miR-25-3p or incubated with exosomal miR-25-3p were transfected with the human KLF2 and KLF4 expressing plasmids (GeneCopoeia). For exosomes transfection, miR-25-3p mimics/inhibitor (GenePharma) were loaded in exosomes using Exo-Fect Exosome Transfection Kit (System Biosciences).

The miR-21-5p mimics, miR-21-5p inhibitor, SIPA1L2 shRNAs and their negative controls (NC-mimics, NC-inhibitor, and sh-NC) were purchased from GenePharma (Shanghai, China). The transfection of miRNAs and SIPA1L2 shRNA into HMECs was performed using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. Transfection of miRNAs into ECFC-exosomes was performed using an Exo-Fect Exosome Transfection Kit (System Biosciences, Palo Alto, USA) according to the manufacturer’s protocol. The sequences of the miR-21-5p mimics, miR-21-5p inhibitor, SIPA1L2 shRNAs and their negative controls are shown in Additional file 1: Table S1.

Antibodies against N-cadherin (22018-1-AP), ZO-1 (21773-1-AP), occludin (13409-1-AP), CD63 (25682-1-AP), CD81 (27855-1-AP), Alix (12422-1-AP), GAPDH (60004-1-Ig), and anti-Ago2 (10686-1-AP) were purchased from Proteintech (Wuhan, China). Antibodies to claudin-5 (343214) and EPB41L5 (614203) were purchased from Zenbio (Chengdu, China). Anti-VE-cadherin antibodies (2500) were obtained from CST (Beverly, MA, USA). Rhodamine B isothiocyanate-dextran was purchased from Sigma-Aldrich (St. Louis, MO, USA). Exo-Fect Exosome Transfection Kit was purchased from System Biosciences (Beijing, China). Recombinant human transforming growth factor β1 (TGF-β1) was purchased from PeproTech (Rocky Hill, USA).

The medium of cultured NSCs was collected every other day and centrifuged at 1500 g for 10 min at 4 °C followed by 10,000 g for 30 min at 4 °C to remove cells, membranes, and debris. The resulting medium was then filtered through 0.22 μm filters. EVs were isolated using the ExoQuick-TC kit (SBI, Mountain View, California, USA). The EVs were confirmed by transmission electron microscopy (TEM). To label the EVs and track the internalization, we transfected EVs with Texas-red labelled RNA oligonucleotides using an Exo-Fect exosome transfection kit (System Bioscience, Palo Alto, CA, USA) following the manufacturer’s instruction.