Figure 3

FNMA by Dunning CaP cells. Immunofluorescence analysis shows that MLL cells cannot assemble a matrix (A), whereas AT-2 (B) and JHU-3 (C) cells can do so to some extent. Neither of the CaP cells, however, can assemble a matrix to the same extent as Rat-2 fibroblasts (D), used here as a positive control. DAPI staining was used to demonstrate equal cell density. Scale bar represents 40 micrometers (A). Biochemical analysis of FNMA by Dunning cells. The assembly of high molecular weight fibronectin multimers (HMWFM) by MLL, AT-2, and JHU-3 cells was assessed using DOC differential solubilization and immunoblot analysis. Actin in the soluble fraction was used as a loading control. The presence of HMWFM is higher in AT-2 and JHU-3 cells than in MLL (B). α5β1 surface expression by Dunning CaP cells. Analysis of normalized MFI for α5β1 integrin expression by the Dunning lines shows that JHU-3 cells express approximately 5-fold more receptors than AT-2 and 7-fold more than MLL. MFI was normalized by subtracting the MFI of the IgG-FITC control histogram from that of the α5β1-integrin specific histogram and expressing net MFI of each cell line relative to that of MLL (C).