Dynasore

Chloroquine was purchased from Wako Pure Chemical Industries (Osaka, Japan). 3-MA, rapamycin, and dynasore were purchased from Cayman Chemical (Ann Arbor, MI). A polyclonal anti-p62 antibody was purchased from Cell Signaling Technology, Inc. (Beverly, MA), a polyclonal anti-β-actin antibody was from Sigma (St. Louis, MO), and a polyclonal anti-LAMP2 antibody was from Bioss Antibodies (Woburn, MA). An inhibitor of cathepsin B (CA-074) was purchased from Peptide Institute, Inc. (Osaka, Japan). Nystatin and cytochalasin D were purchased from Sigma. A monoclonal anti-apoA-I antibody (Wt20-7) was produced as previously described14 (link). pEGFP-N1-TFEB was a gift from Shawn Ferguson (Addgene plasmid #38119)59 (link).

Various antibodies were purchased from the following suppliers: HIV-1 Tat (#ab43014) from Abcam, Cambridge, MA, USA; against phospho-DRP1 (#3455), Alix (#2171), GRP94 (#2104), and p-eNOS (#9571) from Cell Signaling Technology, Danvers MA, USA; against CD81 (#sc-23962), CD63 (#sc-5275), and TSG101 (#sc-7964) from Santa Cruz Biotechnology, Dallas, TX, USA; β-actin (#A5441) from Sigma-Aldrich; total DRP-1 (#611112), total eNOS (#610296) from BD Transduction Laboratory, San Jose, CA, USA. Vybrant DiD cell-labeling dye for exosome staining was purchased from Molecular Probes (Eugene, OR, USA). Dihydroethidium (DHE) (Thermo Fisher Scientific, Waltham, MA, USA) was used to detect cytosolic superoxide and MitoSOX Red (Molecular Probes) for the detection of mitochondrial superoxide. To label mitochondria, MitoTracker Red and MitoTracker Green were used (Molecular Probes). Phorbol 12-myristate 13-acetate (PMA), PX866, Dynasore, and Cytochalasin D were from Cayman Chemical (Ann Arbor, MI), Ionomycin was from Sigma-Aldrich, and recombinant human IL-2 (rIL-2) was from Miltenyi Biotec (Auburn, CA, USA). GW4896 was from Selleckchem (Houston, TX).

FITC-labeled-CTB, and -dextran (MW 70-kDa) were purchased from Sigma and used at final concentrations 10 μg/ml and 3 mg/ml, respectively. Inhibitors of endocytotic pathways, genistein (Wako, Japan), dynasore (Cayman Chemical, MI, US), and latrunculin (Wako), were used at final concentrations, 200, 50, and 2.5 μM, respectively.

GFP-HUVECs were seeded at 200,000 cells per well in a 6-well plate and grown for 48 h. Large EVs (100 µg) isolated from UM cells were labeled with the VybrantTM DiI Cell-Labeling Solution (Invitrogen) for 30 min at 37 °C in the dark. DiI-labeled EVs were purified with PD SpinTrap G-25 columns (Cytiva) following the manufacturer’ protocol. As controls, GFP-HUVECs were pretreated for 24 h with 80 µM Dynasore (Cayman Chemical), or EVs were pretreated for 30 min with 100 µg/mL proteinase K (Invitrogen). At 24 h, large EVs were added to the 6-well plate to be internalized by GFP-HUVECs during 24 h. At 48 h, Matrigel^® layers were deposited into wells of concavity slides (Electron Microscopy Sciences) and incubated for 1 h at 37 °C. GFP-HUVECs (30,000 cells per well in 100 µL) were added onto the Matrigel^® layers and incubated at 37 °C in culture medium with or without angiogenic supplements to perform the endothelial tube formation assay [59 (link)]. Phase contrast images were taken at 2 h, 4 h, 6 h, and 12 h, whereas representative fluorescence images were taken at 6 h. Then, samples were fixed with 4% paraformaldehyde in PBS for 10 min and stained with DAPI (dilution 1:5000; Invitrogen). The characteristics of the pseudo vascular organization of GFP-HUVECs were analyzed using the Angiogenesis Analyzer for ImageJ.