Fig 2: QRT-PCR detect the mRNA expression of HIF-1a, MMP-2, TGF-ß, Twist, N-cad, E-cad in each group of cells (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with Control group; #p<0.05, ##p<0.01, ###p<0.001, ###p<0.0001 compared with shHIF-1a group).

Fig 3: Twist1 interference screening. (A) Representative western blots of Twist1 expression in the different groups. (B) Densitometric analysis of Twist1 expression, as determined by western blotting. siRNA-NC, cells transfected with siRNA-NC for 72 h; siRNA1/2/3-Twist1, cells transfected with siRNA1/2/3-Twist1 for 72 h. Data are presented as the mean ± standard deviation. Data analysis was performed by one-way ANOVA followed by Tukey's post-hoc test. **P<0.01. siRNA, small interfering RNA; NC, negative control.

Fig 4: The HGF/MET signaling pathway activates Nanog expression in CD44v6+ HCC cells. (a) Silencing Met decreased the expression of stemness relative genes, including Oct4, Sox2, and Nanog in CD44v6+ SNU-398 cells. ß-Actin was used as a normalized control. (b) The expression of EMT-related genes, including Snail1, Slug, and Twist in CD44v6+ SNU-398 cells. ß-Actin was used as a normalized control. (c) Western blot showed that silencing Met decreased the expression of Nanog in CD44v6+ cells. But LvNanog did not affect the Met expression. ß-Actin was used as a normalized control. (d) 1 × 105 of cells were injected into the left lobes of the liver. Bioluminescence signals from Met shRNA groups were weaker than that from corresponding control groups. But overexpressed Nanog could rescue the bioluminescence signals. The central luminescence intensity: 8.8e + 7 vs. 1.6e + 6; *p < 0.05, **p < 0.01; t-test. The representative images of Ki67 immunohistochemistry. Scale bar, 50 µm. (e) The representative images of spheres and histogram analysis in indicated cells. Scale bar, 200 µm. (f) and (g) transwell migration and invasion assays showed the knockdown of Met decreased the migration and invasion of CD44v6+ cells, while overexpressed Nanog could rescue the migration and invasion ability. Scale bar, 200 µm. *p < 0.05, **p < 0.01, ***p < 0.001; t-test.

Fig 5: The levels of fatty acid binding protein 4 (FABP4) and IL1a are associated with the phenotype of tumor-associated macrophages (TAMs) and monocytes in neuroblastoma (NB) tissues and blood from different stages, respectively. (A) Representative images of Ki67, Twist, and IL1a immunohistochemistry (IHC) staining in different stages of NB tissues. The average positive ratio from 3 to 5 fields was counted and identified by symbol and color in (B). **p < 0.01. ***p < 0.001. Scale bar represents 50 µm. (C) Representative flow cytometric plots of CD80+CCR7+, CD36+CD209+macrophages in early- and advanced-stage NB tissues. (D) Representative flow cytometric plots of CD14+CD16– monocytes in early- and advanced-stage NB tissues. Data in C–D are presented as the mean ± SD (n = 5). *p < 0.05, **p < 0.01. (E) Concentration of secreted FABP4 in THP1-derived macrophages (THP1-DMs) and human peripheral blood mononuclear cells-derived macrophages (PBMC-DMs) with FABP4 knockdown and overexpression, respectively. Data in E are presented as the mean ± SD (n = 3). ***p < 0.001. (F) Concentration of secreted FABP4 in early-stage (stages I and II) and advanced-stage (stages III and IV) NB samples. (G) Concentration of secreted IL1a in early-stage (stages I and II) and advanced-stage (stages III and IV) NB samples. Data in F–G are presented as the mean ± SD (n = 10). *p< 0.05, **p < 0.01