High density tumor microenvironment co-cultures induce EMT with loss of epithelial markers and increase of mesenchymal markers.

A-C: HCT116 high density mono-cultures were either left untreated (HCT, Co.) or were co-cultured with MRC-5 in monolayer. Tumor microenvironment co-cultures were either left untreated (Co.), treated with curcumin alone (5µM), 5-FU alone (1, 5, and 10µM) or were pretreated for 4 h with curcumin (5µM) followed by treatment with 5-FU (0.1, 1, 2, 3µM). After 10 days of culture, total cell lysates of HCT116 high density cultures were prepared and immunoblotting performed for vimentin (A) or E-cadherin (B) or Slug (C). D-I: HCT116 high density mono-cultures were either left untreated (HCT, Co.) or were co-cultured with MRC-5 in monolayer. Tumor microenvironment co-cultures were either left untreated (Co.), or treated with curcumin (1, 5, 10, 20 µM). After 10 days of culture, total cell lysates of HCT116 HD-cultures were prepared for immunoblotting (D-F) or immunofluorescence performed on sections (G-I) for vimentin (D, G) or E-cadherin (E, H) or Slug (F, I). Densitometric evaluation of protein expression as revealed by western blot analysis was performed in triplicate. Housekeeping protein β-actin served as a loading control in all experiments. Values were compared to the control and statistically significant values with p<0.05. Significant values are marked with (*). F =  Filter. (>) =  HCT116 cells. Magnification G-I: 200×, bar = 30 nm.