FXIII-A activity regulates cytoskeletal dynamics; FN is a major extracellular substrate of FXIII-A. (A) Immunofluorescence microscopy of cell morphology and cytoskeletal elements of preadipocytes upon inhibition of FXIII-A activity with NC9. Inhibition attenuates actin stress fiber formation, promotes cortical actin assembly, and reduces focal adhesion formation (actin and vinculin colocalization). F-actin (red), vinculin (green), and focal adhesions (merge, yellow) (n = 3). Scale bar represents 10 µm. (B) Immunofluorescence microscopy of the dansyl group of NC9 (green) shows its incorporation into FXIII-A enzyme at the periphery of preadipocytes, which is accompanied by disappearance of the actin stress fibers seen in control cells. Actin (red) and DAPI (blue) (n = 3). Scale bar represents 10 µm. (C) WB analysis of MDC-labeled preadipocyte extracts, blotted for dansyl, demonstrates that the substrate probe labels a 250-kDa protein(s) in preadipocyte cultures. Immunoprecipitation with dansyl antibody followed by detection with FN antibody shows the labeled substrate is FN (n = 3). Ig, immunoglobulin. (D) In situ extracellular TG activity assay, ie, analysis of BPA incorporation into coated FN by cellular TG activity. Biotin detection with Neutravidin was performed after all cells were removed. NC9 reduces BPA incorporation into coated FN. ***P < .001. Error bars represent SEM (n = 4). DMSO, dimethylsulfoxide.