Fig. 5

Matrices deposited from both EDA– fibronectin and Snai1 KO MEFs are sensitive to metalloproteinases. A Fibronectin fiber organization in EpRas cells co-cultured with fibroblasts. EpRAs cells and the indicated MEFs were co-cultured on glass coverslips in the presence or absence of 25 µM GM6001 for 3 days. Co-cultures were analyzed by IF with anti-fibronectin (green) and DAPI (blue). Microscopy images are shown. B Fibronectin fiber lacunarity in EpRas co-cultured with fibroblasts is EDA and metalloproteinase dependent. Lacunary in fibronectin images obtained as in A was quantified using the TWOMBLI plugin of ImageJ software. The fold-increase with respect to values in untreated control MEF co-cultures is shown. C Fibronectin fiber organization in HT-29 M6 co-cultured with fibroblasts. Tumor cells and the indicated MEFs were co-cultured on glass coverslips for 6 days. Co-cultures were analyzed by IF with anti-fibronectin (green) and phalloidin (white). Microscopy images are shown. D HT-29 M6 colonies co-cultured with fibroblasts control the presence fibronectin around them in an EDA-dependent manner. For each HT 29 M6 colony, the perimeter and associated empty area (black surface) were quantified (ImageJ software) from images obtained as in A. The fold-increase of the “black area/perimeter” in each co-culture with respect to values in control MEF co-cultures is shown. E The metalloproteinase inhibitor GM6001 rescues the EDA-lacking fibronectin deposition around HT-29 M6 colonies. Cocultures of HT-29 M6 and indicated MEFs were carried out and imaged as in C in the presence or absence of the 25 μM GM6001. Black area measurements and plotting were carried out as in B. Fold increase with respect to values in untreated EDA– MEF co-cultures is shown. F The metalloproteinase inhibitor GM6001 rescues the lack of fibronectin accumulation around HT-29 M6 colonies co-cultured with Snai1 KO MEFs. Co-cultures with indicated cells were established, treated and analyzed as in C and D. The fold-increase with respect to values in untreated control MEF co-cultures is shown