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Fibronectin (Fn) is an extracellular matrix glycoprotein with mechanosensitive structure-function. EDA Fn, a Fn isoform, is not present in adult tissue but is required for tissue repair. Curiously, EDA Fn is linked to both regenerative and fibrotic tissue repair. Given that Fn mechanoregulates cell behavior, Fn EDA organization during wound closure might play a role in mediating these differing responses. One mechanism by which cells sense and respond to their microenvironment is by activating a transcriptional co-activator, Yes-associated protein (YAP). Interestingly, YAP activity is not only required for wound closure, but similarly linked to both regenerative and fibrotic repair. Therefore, this study aims to evaluate how, during normal and fibrotic wound closure, EDA Fn organization might modulate YAP translocation by culturing human dermal fibroblasts on polydimethylsiloxane (PDMS) substrates mimicking normal (soft: 18 kPa) and fibrotic (stiff: 146 kPa) wounded skin. On stiffer substrates mimicking fibrotic wounds, fibroblasts assembled an aligned EDA Fn matrix comprising thinner fibers, suggesting increased microenvironmental tension. To evaluate if cell binding to the EDA domain of Fn was essential to overall matrix organization, fibroblasts were treated with Irigenin, which inhibits binding to the EDA domain within Fn. Blocking adhesion to EDA led to randomly organized EDA Fn matrices with thicker fibers, suggesting reduced microenvironmental tension even during fibrotic wound closure. To evaluate if YAP signaling plays a role in EDA Fn organization, fibroblasts were treated with CA3, which suppresses YAP activity in a dose-dependent manner. Treatment with CA3 also led to randomly organized EDA Fn matrices with thicker fibers, suggesting a potential connected mechanism of reducing tension during fibrotic wound closure. Next, YAP activity was assessed to evaluate the impact of EDA Fn organization. Interestingly, fibroblasts migrating on softer substrates mimicking normal wounds increased YAP activity but on stiffer substrates, decreased YAP activity. When fibroblasts on stiffer substrates were treated with Irigenin or CA3, fibroblasts increased YAP activity. These results suggest there may be disrupted signaling between EDA Fn organization and YAP translocation during fibrotic wound closure that could be restored when reestablishing normal EDA Fn matrix organization to instead drive regenerative wound repair., Competing Interests: Conflict of Interest The authors report no conflicts of interest.