Fig. 2

Solamargine elicits an immunostimulatory tumor microenvironment via macrophages in hepatocellular carcinoma. a Cell viability was exanimated after SM treatment in THP-1 and RAW 264.7 cells. b Co-culture of macrophages and tumor-conditioned medium (TCM) for 24 h to induce M2-like macrophage, then treated with SM for 24 h, the expressions of M2 associated gene (TGF-β, IL-10) and M1 associated gene (INOS, IL-6) were measured. c Macrophages were co-cultured with SM for 24 h, the protein expressions of M1 associated genes were measured. d Macrophage were co-cultured with IL-4 and IL-13 for 24 h to induce M2-like macrophage, then treated with SM for 24 h, the expressions of M2 associated genes were measured by qRT-PCR assay. e The phase-contrast photomicrographs showed the morphology after treatment with SM plus TCM or SM alone. f M2-like macrophages were treated with SM for 24 h, the culture medium was aspirated, and macrophages were co-cultured with HuH-7-GFP cells for 12 h. The phagocytosis of macrophages was detected by FACS analysis. g M2 macrophage were pretreated with SM and placed in the upper chamber to test the invasion ability of HCC cells in the lower chamber. The expressions of LIF, E-cad, N-cad and Vim were determined by western blotting. h HCC cells were treated with or without RSM-CM or TSM-CM. The scratch assay was used to measure migration capabilities of HCC cells. Representative images were shown. i The final tumor weight of H22 subcutaneous tumor mice. j Tumor-associated macrophages, MDSCs, DCs and infiltrating T cells in endpoint tumors were analyzed by Flow cytometry analysis. k Immunofluorescent microscopy images of tissue sections were stained with antibodies against mouse CD206 (green) and F4/80 (red) to observe tumor-associated macrophages. l A schematic diagram illustration of dual synergistic anti-cancer activities of SM. Actin was used as a loading control. Data were presented as means ± SD, ns means no significance, *p < 0.05, **p < 0.01, ***p < 0.001; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001