CAFs secrete and assemble more FN than NAFs. (A) Immunoblot analysis. Conditioned media prepared from NAFs and CAFs from three patients were probed with FN antibody. α-Tubulin served as a loading control. Protein amount is represented by normalizing to α-tubulin. Results are represented as column bars for n = 3 separate experiments. For the FN scale: soluble FN loaded at a range of 300 to 0.75 ng. (B, left) Immunostaining of FN (green) in NAFs and CAFs from three patients. F-actin is stained with phalloidin-rhodamine (red), and DNA was stained with DAPI (blue). Bar, 20 µm. (Right) Quantification of assembled FN. Amount of assembled FN is defined as the amount of fluorescence in a cell (integrated density) normalized to the area of the cell and the background fluorescence. P-value is calculated using Mann–Whitney test for n = 20 cells over n = 2 separate experiments. (C) Scatter dot graphs correlating the invasion index of cancer cells in the presence of fibroblasts from all three patients with the amount of secreted (left) and assembled FN (right) by fibroblasts. Quality of linear regression is represented by the values of p and r². (D) Quantification of cancer cell invasion in a collagen matrix or in a collagen and FN matrix. Invasion index is defined as the ratio between the number of invading nuclei of GFP cancer cells and the area of the spheroid contour. Quantification results are expressed as box and whiskers (minimum to maximum) for at least n = 3 separate experiments. ***, P < 0.001. A.U., arbitrary units.