Figure 3

M2 polarized macrophages induce trans-differentiation, activation of the β-catenin pathway and invasion of F9- and NMuMG cells in vitro. (A) Immunofluorescence of E-cadherin, β-catenin, vimentin and fibronectin expression and cellular localization in F9-cells cultured in F9-CM or M-CM for 24 h or 7d. Pictures of NMuMG-cells are shown in Additional file 2: Figure S1. Scale bar = 0.02 mm. Proteins are colored red and nuclei were stained with DAPI (blue). Arrows indicate the location of the annotated protein in cells cultured in M-CM for 7d. (B) Relative protein expression normalized to β-actin levels (quantified from western blots shown in Additional file 2: Figure S2.) of E-cadherin, total and active β-catenin in F9-cells (left panels) and NMuMG-cells (right panels) cultured in F9-CM, N-CM or M-CM for 6 h, 24 h, 5d, 7d, and 13d. (C) Immunofluorescence of E-cadherin and β-catenin expression and cellular localization in F9-cells cultured in M-CM for 7d (left panel), followed by additional culture in F9-CM for 7d (right panel). Scale bar = 0.02 mm. Proteins are colored red and nuclei were stained with DAPI (blue). Arrows indicate the location of the annotated protein in cells after 7d culture in F9-CM. (D) Relative protein expression normalized to β-actin levels (quantified from western blot shown in Additional file 2: Figure S2A) of E-cadherin, total and active β-catenin in F9-cells cultured in M-CM for 7d, followed by F9-CM for additional 24 h and 7d. (E) Fold luciferase expression (TOPFLASH/FOPFLASH) in F9-cells (left) and NMuMG-cells (right) after 7d or 13d of culture, respectively, in F9-CM, N-CM or M-CM; n = 7; bar = means ± SEM; *P < 0.05. **P < 0.005, unpaired t-test. (F) Invasion by F9-cells (n = 8) and NMuMG-cells (n = 3) in response to F9-CM, N-CM or M-CM (48 h, 1% Matrigel) calculated relative to F9-CM or N-CM, respectively. **P < 0.005, unpaired t-test; bars = means ± SEM.