Images of immunostained ZO-1, VE-cadherin, and claudin-5 were analyzed using the JAnaP to calculate the percent of the cell perimeter presenting continuous, punctate, or perpendicular junctions. using the JAnaP to calculate the percent of the cell perimeter presenting continuous, punctate, or perpendicular junctions. Transwell permeability assays and resistance measurements were used to measure bulk (global) barrier properties, and a local permeability assay was used to correlate junction presentation proximal to permeable monolayer regions. Results Substrate composition was found to play little role in junction presentation, while cAMP supplements significantly increased the continuous junction architecture. Increased culture time required increased cAMP treatment time to reach comparable ZO-1 and VE-cadherin protection observed with shorter culture, though longer cultures were required for claudin-5 presentation. Continuous cAMP treatment (6?days) disrupted junction integrity for all those three junction proteins. Transwell permeability and TEER assays showed no correlation with junction phenotype, but a local permeability assay revealed a correlation between the S38093 HCl quantity of discontinuous and no junction regions with barrier penetration. Conclusions These results suggest that cAMP signaling influences HBMEC junction architecture more than matrix composition. Our studies emphasized the need for local barrier measurement to mechanistically understand the role of junction S38093 HCl phenotype and supported previous results that continuous junctions are indicative of a more mature/stable endothelial MTC1 barrier. Understanding what conditions influence junction presentations, and how they, in turn, affect barrier integrity, could lead to the development of therapeutics for diseases associated with BBB dysfunction. suggest that increased continuous junction presentation is associated with a less permeable barrier, with increased gaps or discontinuous junctions indicating increased permeability. Our methods could provide useful quantitative insights into time-dependent changes in junction architecture that occur in different biochemical or mechanical conditions. Understanding what conditions influence junction presentations and how that affects barrier properties could lead to therapeutic development S38093 HCl for diseases associated with BBB dysfunction or delivery mechanisms capable of traversing healthy barrier systems. Conclusion In summary, we investigated the influence of cell culture parameters such as matrix protein covering, culture time, and cAMP treatment, and used the JAnaP to quantify their role in cell and junction morphology. While protein covering seemed to have only a modest effect on these parameters, cAMP treatment significantly increased continuous junction presentation. Total cell culture time did not increase junction presentation, but instead required increased cAMP treatment for protein protection comparable to shorter culture time. No correlation between junction presentation and barrier permeability was found when comparing junction phenotype to Transwell-based TEER and permeability experiments, motivating the use of an assay that could instead capture cell-to-cell inhomogeneities rather than a bulk barrier measurement. A local permeability assay recognized that barrier permeability most closely correlates with the number of gaps with no junction protection, and by extension, the number of discontinuous junctions, present at the cell edge. Together this promotes the use of local measurement techniques to quantitatively study barrier function in conjunction with junction phenotype to investigate the mechanisms at play in functional and dysfunctional barrier systems. Supplementary information Additional file 1. This Additional?file contains Figures S1-S16,?Furniture S1-S7, and Additional?Method S1.(9.1M, pdf) Acknowledgements The authors would like to acknowledge Kyle Thomas S38093 HCl at Yellow Basket, LLC (kyle@yellowbas- ket.io) for JAnaP software development support, and the University or college of Maryland. Abbreviations BBBBloodCbrain barrierB-FBNBiotinylated fibronectinCNCollagen ICPT-cAMP8-(4-Chlorophenylthio) adenosine-3,5-cyclic monophosphate sodium saltCIVCollagen IVECsEndothelial cellsFBNFibronectin F:C:Lfibronectin?+?collagen IV?+?lamininHBMECHuman brain microvascular endothelial cellHUVECHuman umbilical vein endothelial cellJANaPJunction Analyzer ProgramLNLamininP_appApparent permeability coefficientPBSDulbeccos Phosphate-Buffered Saline containing calcium and magnesiumPRPermeated regionRO-20-17244-(3-Butoxy-4-methoxybenzyl) imidazolidin-2-oneTEERTransendothelial electrical resistanceVE-cadherinVascular endothelial cadherinZO-1Zonula occludens 1 Authors contributions KMG and KMS designed the research and wrote the manuscript. JWJ performed TEER assay measurements and aided in JAnaP analysis. CTI performed?transwell permeability assay measurements. KMG performed immunostaining, microscopy, and all other experiments. All authors read and approved the final manuscript. Funding The authors acknowledge funding S38093 HCl from your Burroughs Wellcome Career Award at the Scientific Interface (to KMS), the Fischell Fellowship in Biomedical Engineering (to KMG), the ASPIRE.