FN assembly by MKs adherent to type I collagen. The ability of MKs to assembly pFN was evaluated on type I collagen and fibrinogen, as described in “Methods.” (A) Assembly of FITC-labeled FN (green) by CD61⁺ MKs (red) adherent on type I collagen and fibrinogen. Scale bars = 10 μm (top), 50 μm (middle) and 30 μm (bottom). In parallel experiments, staining of fibrillar pFN with a polyclonal antibody in MKs plated on type I collagen and fibrinogen. MKs adherent on type I collagen and fibrinogen were removed after DOC treatment, and DOC-insoluble pFN fibrils were stained with a polyclonal antibody anti–FN (red). Nuclei were always counterstained with Hoechst 33288 (blue). (B) The presence of DOC-insoluble assembly of pFN was also revealed by immunoblot analysis using a monoclonal anti–FN, clone IST-4. In a parallel sample, cells were scraped and proteins separated by SDS-PAGE and then stained with anti–β actin to ensure the same number of cells in the experiment. (C) Evaluation of cytoskeleton and integrin roles in FN matrix assembly by MKs in samples treated with RGDS or blebbistatin with respect to controls. FN matrix after DOC treatment was stained with the polyclonal antibody anti–FN and visualized in immunofluorescent dye. (D) The presence of endogenous EDA⁺ FN during pFN deposition and assembly was evaluated with immunofluorescent dye, and staining was performed using both the polyclonal antibody against FN and monoclonal anti–EDA FN. Scale bars = 20 μm (C-D).