Fig. 6. USF-1 in situ localization and PAI-1 E-box-binding activity in wound-proximal keratinocytes. Compared with quiescent intact monolayer regions (A), HaCaT cells juxtaposed to the wound site (arrow in B indicates direction of migration into a denuded zone) exhibit significant cytoplasmic and nuclear immunocytochemical reactivity for USF-1 (B). In both panels A and B, nuclei are stained (red) with propidium iodide while green speckles indicate immunoreactive USF-1. Cells situated more distal (d) from the wound edge had considerably lower cytoplasmic and nuclear USF-1 (B). Nuclear USF-1 accumulation could be detected as early as 2 hours post-monolayer scraping (preceeding the increase in PAI-1 transcripts) and remained evident throughout the period of wound repair. The protracted (48-72 hour) time course of injury resolution in HaCaT cultures reflected (even at 24 hours after wounding) continued PAI-1 expression (Fig. 1) and nuclear USF-1 localization (B) by the migrating epithelium. The typical upper and lower dumbell-shaped gel retardation pattern was resolved upon incubation of nuclear extracts from growing (G), but not quiescent (Q), HaCaT and RK cultures with the ³²P-labeled 18 bp PAI-1 E-box probe (C). The upper band was specifically supershifted upon addition of antibodies to USF-1 after formation of the protein-probe complex. Similarly, the USF-1-containing upper complex was also resolved upon incubation of nuclear extracts from RK cells harvested from the wound site (D). The upper-lower doublet retardation pattern was evident as early as 2 hours after scrape injury (2 hr edge) and, like growing RK cultures (G), addition of USF-1 antibodies specifically supershifted this upper complex.