Western blotting and exosome uptake

Exosomal vesicles derived from U937 cells and macrophages were lysed in RIPA lysis buffer. Antibodies against FAM53B (1:1000, A19236; Abclonal, Wuhan, China), GAPDH (1:2000, AC001; Abclonal) and Tubulin were used. Protein samples were adjusted to 0.375 mg/mL, 0.75 mg/mL, 1.5 mg/mL, 3 mg/mL, and Graphpad software (San Diego, CA, United States) was used to generate a standard protein curve. After washing, the membranes were incubated with peroxidase-labeled goat anti-rabbit secondary antibody (1:2000, AS014; Abclonal) at 37 °C for 1 h. The PKH67 Fluorescent Cell Linker Kit was used to identify and stain exosome suspensions. Diluent C (250 μL) was added to 50 μL of exosomes, followed by 1.5 µL diluent C (1.5 μL). The labeled and diluted exosomes were completely suspended with DMEM, and the exosomes were then added to the U937 cell (DAPI staining) culture supernatant with a pipette. After 24 h of culture, fixation, membrane rupture, and nucleation, laser scanning confocal fluorescence microscopy was used to observe whether U937 cells could take up macrophage-derived exosomes.

The gray value was detected by Image J software (version 1.8.0; National Institutes of Health, Bethesda, MA, United States), and the results were calculated by Graphpad Prism (version 9.0; GraphPad Software, San Diego, CA, United States).