100 kda filter

As indicated in previous reports [36 (link)], LDL fractions were diluted to a protein concentration of 0.2 mg/mL with phosphate buffer (50 mM PBS, pH 7.4); copper sulfate was added to a final concentration of 0.06 mM and incubated for 2, 8, and 24 h in a thermostatic chamber at 37 °C. Oxidation was stopped by adding ethylenediaminetetraacetic acid (EDTA) to a final concentration of 1.0 mM. To prevent oxidation due to residual copper ions, the solvent was replaced with phosphate buffer (50 mM PBS, pH 7.4) using a 100 kDa filter (Merck Millipore Ltd., Cork, Ireland) [37 (link)]. Oxidized LDL solutions were diluted to a protein concentration of 0.2 mg/mL and stored at 4 °C until immediately before use. nLDL was added to equal volumes of water instead of copper sulfate. As with oxLDL, EDTA was added and stored at 4 °C until use.

EVs isolated from MCF-7 and MCF-7Cx46-GFP cells respectively, were loaded with a plasmid encoding green fluorescent protein (GFP) according to the protocol published by Soares. A et al. [20 ] with a few modifications. Briefly, 5 μg of plasmid were mixed with 10 μg of Lipofectamine 2000 (Life Technologies) in Opti-MEM for 10 min; then 25 μg of EVs were incorporated to the mix and incubated for 30 min at room temperature. To eliminate the non-incorporated plasmid, 5 U of DNAse I (New England Biolabs) were added and incubated for 30 min at 37 °C. EVs were filtered through a 100-kDa filter (Millipore, Burlington, USA). Then, 5 × 10⁵ of MCF-7 recipient cells were seeded on 35 mm dishes, 24 h later 2.5 μg of DNA-loaded EVs-sansCx46 or EVs-Cx46 were co-cultured with recipient cells for 45 min. After that, the medium was replaced with EVs-depleted medium and left for 24 h to analyze protein expression. GFP expression was evaluated by flow cytometry and fluorescence microscopy. For flow cytometry, MCF-7 cells were detached with trypsin, rinsed with PBS three times and resuspended in PBS. Cytometry was performed using a FACSCantoII (BD Biosiences, Franklin Lakes, USA). For fluorescent microscopy, cells were fixed with 4% PFA, mounted with Fluoromount (Sigma-Aldrich, Saint Louis, MO, USA) and visualized using a Nikon Eclipse Ti-U inverted microscope (Nikon, Melville, NY, USA).

HEK293T cells were cultured on six 100-mm plates and transfected with 6 µg recombinant plasmids per dish using the Lipofectamine 2000 reagents (Invitrogen). After being maintained for 48 h at 5% CO₂ and 37 °C, the cells were collected, resuspended in 6 ml PBS, and lysed by subjecting to four rounds of freezing (−80 °C) and thawing (37 °C). Benzonase (Novagen) was added to the mixture (50 U/ml, final concentration) and incubated for 30 min at 37 °C. The cell lysate was then spun at 10,000 rpm for 30 min. The supernatant was loaded into a 13-ml centrifuge tube with a continuous CsCl gradient and spun in a SW41 rotor (Beckman) at 36,000 rpm for 36 h. The recombinant virus was collected and desalted by centrifugation through the 100 KDa filter (Millipore) according to the manufacturer’s instructions. Viral DNA was extracted with the TIANamp Virus DNA/RNA Kit (TIANGEN, Beijing, China) according to the manufacturer’s protocol.