Exploratory experiment designed to discover new patterns targeting CDH5 in lymphatic endothelial cells and animal models. Primary outcome: demonstration of VE-cadherin requirement for reelin secretion
This experiment investigated the novel finding that VE-cadherin plays a requisite role in controlling the secretion of reelin, a lymphangiocrine glycoprotein. The researchers conducted both in vitro cell culture experiments and in vivo studies to demonstrate this regulatory relationship. Reelin is a recently appreciated factor with important roles in governing heart development and injury repair. The experiments likely involved measuring reelin secretion levels under different conditions of VE-cadherin expression or function, potentially using techniques such as ELISA, Western blotting, or immunofluorescence. The in vivo component may have involved animal models to validate the physiological relevance of this regulatory mechanism.
VE-cadherin Knockdown: Transfect subconfluent HMVEC-LLy with VE-cadherin (CDH5) siRNA (Dharmacon #J-011459-09-0005, 50 nM) or non-targeting control siRNA (D-001810-10-05) using RNAiMAX (Life Technologies). Knockdown efficiency assessed at 48h and 72h via qRT-PCR (target: ≥75% mRNA reduction) and western blot (target: ≥60% protein reduction).
In Vivo Confirmation: VE-cadherin deletion in adult mice must produce ≥30% reduction in lymphatic Reelin content and altered lymphatic function (either reduced contractile frequency or impaired response to shear stress) within 4 weeks of induction.
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