Sustained intestinal epithelial monolayer wound closure after transient application of a FAK-activating small molecule

M64HCl, which has drug-like properties, is a water-soluble Focal Adhesion Kinase (FAK) activator that promotes murine mucosal healing after ischemic or NSAID-induced injury. Since M64HCl has a short plasma half-life in vivo (less than two hours), it has been administered as a continuous infusion with osmotic minipumps in previous animal studies. However, the effects of more transient exposure to M64HCl on monolayer wound closure remained unclear. Herein, we compared the effects of shorter M64HCl treatment in vitro to continuous treatment for 24 hours on monolayer wound closure. We then investigated how long FAK activation and downstream ERK1/2 activation persist after two hours of M64HCl treatment in Caco-2 cells. M64HCl concentrations immediately after washing measured by mass spectrometry confirmed that M64HCl had been completely removed from the medium while intracellular concentrations had been reduced by 95%. Three-hour and four-hour M64HCl (100 nM) treatment promoted epithelial sh..., , , ***Sustained intestinal epithelial monolayer wound closure after transient application of a FAK-activating small molecule*** Access this dataset on Dryad:Â Â [https://doi.org/10.5061/dryad.qnk98sfqp](https://doi.org/10.5061/dryad.qnk98sfqp) We compared the effects of shorter M64HCl treatment in vitro to continuous treatment for 24 hours on monolayer wound closure. We then investigated how long FAK activation and downstream ERK1/2 activation persist after two hours of M64HCl treatment in Caco-2 cells. M64HCl concentrations immediately after washing measured by mass spectrometry confirmed that M64HCl had been completely removed from the medium while intracellular concentrations had been reduced by 95%. Three-hour and four-hour M64HCl (100 nM) treatment promoted epithelial sheet migration over 24 hours similar to continuous 24-hour exposure. 100nM M64HCl did not increase cell number. Exposing cells twice with 2-hr exposures of M64HCl during a 24-hour period had a similar effect. Both FAK i...M64HCl是一种具备类药特性的水溶性黏着斑激酶（Focal Adhesion Kinase, FAK）激活剂，可促进缺血或非甾体抗炎药（NSAID）诱导损伤后的小鼠黏膜愈合。由于M64HCl在体内的血浆半衰期较短（不足2小时），既往动物研究中均采用渗透微型泵进行持续给药。然而，短暂暴露于M64HCl对单层细胞伤口闭合的影响仍不明确。本研究首先在体外比较了短时M64HCl处理与24小时持续处理对单层细胞伤口闭合的效果；随后在Caco-2细胞中探究了M64HCl处理2小时后，FAK激活及下游ERK1/2激活的持续时长。通过质谱法检测清洗后即刻的M64HCl浓度，证实培养基中M64HCl已完全清除，而细胞内浓度降低了95%。3小时和4小时的100 nM M64HCl处理可促进上皮细胞迁移……**短暂应用黏着斑激酶激活小分子后持续促进肠上皮单层伤口闭合**可在Dryad数据库获取本数据集：https://doi.org/10.5061/dryad.qnk98sfqp本研究首先在体外比较了短时M64HCl处理与24小时持续处理对单层细胞伤口闭合的效果；随后在Caco-2细胞中探究了M64HCl处理2小时后，FAK激活及下游ERK1/2激活的持续时长。通过质谱法检测清洗后即刻的M64HCl浓度，证实培养基中M64HCl已完全清除，而细胞内浓度降低了95%。3小时和4小时的100 nM M64HCl处理可在24小时内促进上皮层迁移，效果与24小时持续暴露相当。100 nM M64HCl不会增加细胞数量。24小时内分两次给予2小时的M64HCl处理，也可达到相似效果。FAK……