Fig. 1. Monolayer injury stimulates PAI-1 synthesis specifically in wound-edge cells. Quiescent contact-inhibited cultures of RK, HaCaT and EC-1 cells were maintained either intact or scrape-wounded with a pipette tip. RK cultures were fixed and PAI-1 protein visualized by immunocytochemistry (A,B). Control quiescent RK cells expressed relatively low levels of PAI-1 (A). Within 6 hours after scraping, PAI-1 was readily detected in motile RK cells immediately juxtaposed to the injury site (B; arrow indicates direction of migration into the denuded area). Western analysis of lysates of HaCaT cells differentially harvested 24 hours after scrape-trauma confirmed a significant increase in PAI-1 expression by epithelial cells bordering the injury site compared with cells in the distal uninvolved monolayer (C). PAI-1 mRNA transcripts were upregulated in cells harvested from the wound edge within 5 hours after scrape-injury. PAI-1 mRNA abundance (normalized to A-50 and GAPD hydridization signal for EC-1/RK and HaCaT cells, respectively) remained elevated over the time course of wound repair (D). RK total RNA was not isolated at the 7 hour post-wounding time point in the analysis series summarized in D. The difference in PAI-1 mRNA kinetic profiles for HaCaT versus EC-1/RK cells reflects the relatively protracted time frame for HaCaT monolayer injury site closure compared with RK/EC-1 populations (i.e. 48-72 hours versus 24-36 hours).

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