Fibronectin matrix assembly is required for vascular basement membrane formation during EC-pericyte tube coassembly. EC-pericyte cocultures were allowed to assemble for 5 days in a 3D collagen matrix either in the presence or absence of 50 μg/mL 70-kDa fragment of fibronectin (using HUVECs-bovine pericytes). (A) Immunofluorescent staining of fibronectin, collagen IV, and laminin demonstrates that disruption of fibronectin assembly leads to disrupted collagen IV assembly (first column in red, with the second column showing overlays denoting the position of GFP-pericytes). (B) Quantification of average vessel width reveals that blockade of fibronectin assembly leads to increased vessel width of EC tubes in the cocultures, but not EC-only cultures (P < .01). (C) Intensity mapping of representative fibronectin and collagen IV stains is shown to demonstrate the reduced levels of assembly/deposition of these proteins.