Fig. 3: Gene silencing by FSGG/siGal-9 in vitro and biological functions of exosome/siGAL-9.

Gene silencing efficiency using FSGG/siGal-9 in vitro. The mRNA expressions of Gal-9 and GAPDH were quantified with RT-q-PCR (a), or by western blots (b) and (c). d Changes of exosome concentration released by B16-F10 cells at different culture temperature 37 °C or 44 °C for 30 min. The concentration was quantified with protein levels which was examined by the BCA method. e, f Exosome and exosome/siGal-9 stimulating tumor-specific splenic lymphocyte proliferation. Exosome was collected from the culture medium of B16-F10 cells, while exosome/siGal-9 was collected from the culture medium of Gal-9-silenced B16-F10 cells. Antigen (Ag) was directly prepared from B16-F10 cells via the frozen-thawed method. The BCA method was used to quantify the concentration of Ag, exosome and exosome/siGal-9. The same amount (50 μg/ml) of Ag, exosome or exosome/siGal-9 was added into the cultured tumor-specific splenic lymphocytes for 24 h. Then the CCK-8 method was used to detect the proliferation of lymphocytes. g Proteomics of exosome or exosome/siGal-9 were analyzed by LC-MS/MS, and the main biological function changes of exosome/siGal-9 were depicted according to the KEGG pathway where bars were labeled with P value. For the KEGG pathway, the crit-up ratio is ≥1.2 and the crit-down ratio is ≤1/1.2. In total, 379 proteins were up and 746 proteins were down. Error bars represent the standard deviation of 3 experiments (*P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001).