Fig. 3.

The activation of Wnt2b/β-catenin signalling enhances TAMs-induced tumour-promoting effects. a THP-1 derived macrophages (THP-1-M) were transfected with control vectors or Wnt2B-V5 (over-Wnt2b) vectors for 48 h. These macrophages were incubated with RPMI 1640 for an additional 24 h to obtain the condition medium (CM). HCC cells were cultured in the presence of indicated CM for 48 h. The expression levels of EMT markers were determined by western blotting. (b-e) THP-1-M infected with control vectors, sh-Wnt2b or sh-CTNNB1 (β-catenin) vectors were acquired as described in Materials and Methods. These macrophages were incubated with 50% HCC-TCM for 48 h for the preparation of the different TAMs. These TAMs were incubated with RPMI 1640 for another 24 h to obtain the CM. b, c HCC cells were cultured in the presence of the indicated CM for 48 h. The expression levels of EMT markers were determined by western blotting. d HCC cells were incubated with culture medium (Ctrl) or the indicated CM for 24 h. The cell viability of each group was detected by MTT assay. e HCC cells were scratched with a plastic pipette tip and incubated with culture medium (Ctrl) or indicated CM for 24 h. The results of this wound healing assay were photographed and measured. One representative of at least three independent experiments is shown. qPCR, quantitative real-time PCR; HCC, hepatocellular carcinoma; TCM, tumour condition culture medium; TAMs, tumour-associated macrophages; 7721, SMMC-7721. Data are presented as mean ± SEM from at least three independent experiments (*p < 0.05, **p < 0.01 and ***p < 0.001)