Enhanced chemiluminescence kit

Total proteins were isolated from cells by using a total protein extraction kit (Vazyme, Nanjing, China), and they were quantified by using a BCA protein assay kit (Vazyme). The proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (BD). The membrane was blocked with 5% skim milk for 3 h and incubated with specific primary antibodies, including anti-SMP-30 (1:200; Abcam, Cambridge, England) and anti-β-actin (1:5000; Fdbio, Hangzhou, China) overnight at 4°C. Then, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (1:5000; Fdbio) for 2 h at 25°C. The protein bands were visualized by using an enhanced chemiluminescence kit (Fdbio) and quantified by using a gel imaging analysis system (Olympus, Tokyo, Japan).

After treatment with siRNA negative control (si-NC), si-Spry3, agomiR-NC, agomiR-18a, agomiR-BART10-5p, agomiR-18a combined agomiR-BART10-5p, antagomiR-NC, antagomiR-18a, antagomiR-BART10-5p, and antagomiR-18a combined antagomiR-BART10-5p, HONE1 and HONE1-EBV cells were harvested and lysed in lysis buffer supplemented with protease inhibitors. MicroRNA mimics, agomiRs, inhibitors, and antagomiRs were transfected at 50 nmol/L with Lipofectamine 2000 reagent (Invitrogen, USA). Protein lysate was resolved on 10% SDS-PAGE followed by blot transfer onto a polyvinylidene fluoride (PVDF) membrane (Millipore, USA) using the semidry NovaBlot system (Amersham Pharmacia, UK) for western blot analysis. After that, the membranes were first incubated with antibodies against GAPDH (Proteintech, USA), Spry3, Ras, c-Raf, MEK1/2, Erk1/2, mTOR, eIF4E1, VEGF, mmp2, and HIF1-α (Abcam, UK) overnight at 4°C, followed by a 1- to 2-h incubation with horseradish peroxidase-conjugated secondary antibody. The protein signals were detected using an enhanced chemiluminescence kit (Fdbio Science, China) and analyzed using the Bio-Rad (USA) imaging system and the associated software according to the manufacturer’s instructions. Antibodies concentrations are listed in Table S1.

A protein extraction reagent (Thermo, USA) was used, followed by centrifugation at 12,000 rpm for 15 min to isolate the protein from cells or exosomes. The protein concentration was determined using a bicinchoninic acid assay kit (Thermo, USA) before Western blotting. In the second step, the proteins were transferred onto 0.2 μm polyvinylidene difluoride membranes (Bio-Rad, USA) for 90 min at 300 mA. After blocking with 5% skim milk Tris-buffered saline with 0.1% Tween^® 20 Detergent (FDbio, China) for 1 h, we probed the target protein by incubating the membranes overnight at 4°C with primary antibodies. Following this, a secondary antibody was incubated for an hour at room temperature on the membranes. The bands were visualised using an enhanced chemiluminescence kit (Fdbio, China), and the band densities were analysed using ImageJ. Matrix metallopeptidase 9 (MMP-9) (Abcam, USA), NFATc1 (Abcam, USA), Runx2 (Huabio, China), STAT3 (Huabio, China), phosphorylated (p)-STAT3 (Huabio, China), JAK2 (Huabio, China), p-JAK2 (CST, USA), CD9 (Abcam, USA), Alix (Huabio, China), CD63 (Abcam, USA), CD81 (Abcam, USA), and GAPDH (Proteintech, USA) were prepared using primary antibody dilution buffer (1:1000, Beyotime, China). Secondary antibodies (Yeason, China) were prepared using 5% skim milk (1:5000).