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Fig. 8. Stimulatory effects of 3D matrix on integrin activities. (A) HT1080 cells were plated on substrates in the presence of 20 µg/ml rat pFN. After 15 hours, 1 mM MnCl2 was added to one set of cells (+). After an additional 4-hour incubation, DOC-insoluble samples were prepared and analyzed by immunoblotting with IC3. (B) CHO(B2){alpha}vß3 or CHO(B2) cells were allowed to attach for 2 hours on substrates prepared from WI-38 (VA13) cells (3D) or coated with human pFN (2D). Rat pFN was added at the indicated concentrations followed by incubation for another 15 hours. The DOC-insoluble fractions were analyzed by immunoblotting with IC3 antibody against rat fibronectin. (C) CHO(B2){alpha}vß3 cells were plated as described in B but with 40 µg/ml rat pFN for 27 hours followed by an additional 4 hours with 1 mM MnCl2. The DOC-insoluble samples were prepared as in B. (D) CHO(B2){alpha}5 and CHO(B2){alpha}vß3 cells were allowed to attach to 3D fibronectin matrices for 1 hour. 1 µg/ml SAM-1 anti-{alpha}5 function-blocking antibody or 10 µg/ml LM609 anti-{alpha}vß3 function-blocking antibody were then added to the cells with 10 µg/ml rat fibronectin (+). After incubation for 16 hours, the DOC-insoluble samples were collected and analyzed by immunoblotting with IC3 antibody.