M2 macrophages regulate EMT through paracrine TGF-β signaling. (A) Immunofluorescence of E-cadherin, β-catenin, vimentin and fibronectin expression and cellular localization in F9-cells cultured in F9-CM, M-CM, M-CM neutralized for TGF-β and in M-CM plus an IgG1 control antibody for 7d. Pictures of NMuMG-cells are shown in Additional file 2: Figure S5. 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 and M-CM + TGF-β neutralizing antibody for 7d. (B) Relative luciferase expression (TOPFLASH/FOPFLASH) in F9-cells (left) and NMuMG-cells (right) after 7d or 13d of culture in F9-CM, N-CM, M-CM or M-CM neutralized for TGF-β, respectively; n = 4-7; bar = means ± SEM. *P < 0.05, unpaired t-test. (C) Relative invasion by F9-cells (left) and NMuMG-cells (right) in response to F9-CM, N-CM, M-CM, M-CM neutralized for TGF-β (TGF-βn), or to M-CM plus an IgG1 control antibody (48h, 1% Matrigel); n = 3-8. Fold invasion was calculated relative to F9-CM or N-CM. *P < 0.05, **P < 0.005, unpaired t-test, bar = means ± SEM.