Figure 3.

Secretion of cytokines by differently derived fibroblasts (H-CAFs and NSFs). (A) A liquid chip assay analysis indicated that the concentrations of these cytokines (IL-6, IL-8, MCP-3, HGF and VEGF) were significantly higher in the CM of H-CAFs than in the CM of NSFs. However, NSFs secreted a higher level of these cytokines (SDF1, MMP2 and MMP3) than the other three fibroblast types. TGF-β secretion was similar among the four fibroblast types. (B) The levels of IL-6 and HGF in the H-CAF CM were further verified by ELISA. (C) RT-PCR was used to detect the expression of IL-6 and HGF in H-CAFs. (D) Recombinant IL-6, HGF or IL-8 was added to treat HCC cells with a dose dependent manner. Indicated antibodies were used to detect the protein level E-cadherin or Slug. (E) Left panel, the corresponding neutralizing antibodies were added to medium after HCC cells were treated with CAF-CM. Right panel, HCC cells were treated with recombinant IL-6, HGF or IL-8. Indicated antibodies were used to test the signals E-cadherin, Fibronectin, Slug, pSTAT3-S727 and STAT3. (F and G) Representative images and analysis show that the IL-6 significantly induced Huh7 cells invasion in vitro. Original magnification, 200×. The error bars represent ± SEM, * P<0.05; ** P<0.01.