L 80xp

The MP was extracted by the method as reported by Zhao et al. (2022) . Briefly, the ground meat (100 g) were homogenized with 4 times volume (w/v) of isolation buffer (0.1 M NaCl, 2 mM MgCl₂, 1 mM (EGTA), 10 mM KH2PO4/K₂HPO₄, pH 7.0) by a homogenizer (T25, IKA, Germany) at 10000 × g for 1 min. Then, the mixtures were centrifuged (1500 ×g, 4 °C) for 0.5 h and the precipitates were harvested. Subsequently, the precipitates were resuspended with 0.1 mol/L NaCl solution at a solid-liquid ratio of 1:4 (g/mL) and then centrifuged (150,000 ×g, 4 °C) for 0.5 h (L-80-XP, Beckman, USA). The harvested precipitates were crude MPs and stored at 4 °C until further analysis (within 48 h). PIPES buffer (15 mM, 0.3 M NaCl) containing 0, 20, 40, 60, 80, and 100 mM CaCl₂, was used for diluting the MP suspensions, respectively.

The MP suspensions were diluted to 5 mg/mL by PIPES buffer (15 mM, pH 6.25) and centrifuged at 8000 ×g for 20 min (L-80-XP, Beckman, USA) and the solubility was measured using the Coomassie brilliant blue method for evaluating the protein content (Wang, Xia, Zhou, Wang, et al., 2020b ). The solubility was defined as follows: $\mathit{Solubility}\left(\%\right)=\frac{\text{Protein content of supernatant}}{5\mathrm{mg}/\mathrm{mL}}\times 100\%$

Exosomes were purified from the plasma of patients with HCC by ultracentrifugation. The plasma was thawed on ice and centrifuged at 500 × g for 10 min and the supernatant was collected. The following steps were completed continuously and without interruption as soon as possible. The supernatant was ultracentrifuged using a W32Ti rotor (L-80XP; Beckman Coulter, Inc.) at 16,800 × g for 30 min to pellet the exosomes. Subsequently, the pellet was washed in PBS to eliminate contaminating proteins, and centrifuged again at 110,000 × g for 70 min. PBS was subsequently removed and the exosomes were re-suspended in 100 µl PBS. All centrifugation steps were performed at 4°C. All samples were stored in a −80°C ultra-low temperature refrigerator until use (within 1 month).

Prior to cell culture, DMEM containing 20% FBS was centrifuged at 120,000 g for 2 h to deplete serum exosomes. HKT293T cells, used for exosome production, were cultured in 30 ml 5% exosome-depleted FBS in a 150 mm dish and maintained in 5% CO₂ at 37°C for 48 h. Exosomes were isolated from the 30 mL harvested supernatant according to a previous report 21 . Briefly, the supernatant was centrifuged at 300 g for 10 min, 1200 g for 20 min, and 10,000 g for 30 min at 4°C to remove cells and cellular debris and then filtered through a 0.22 μm filter (Millipore, Billerica, MA, USA). The filtrate was centrifuged at 110,000 g for 120 min at 4°C in a Type Ti70 rotor, using an L-80XP ultracentrifuge (Beckman Coulter, Brea, CA, USA). The exosome pellet was resuspended in PBS and ultracentrifuged again at 110,000 g for 120 min 22 . The pelleted exosomes were resuspended in PBS and analyzed using a Micro BCA Protein Assay kit (Pierce, Rockford, IL, USA) or by western blotting analysis of exosomal markers using antibodies specific for Alix, Tsg101, and endoplasmic reticulum marker GM130 (Proteintech, Wuhan, China).