FIG. 5.

Analysis of DNA synthesis in organotypic raft cultures. HPV16 reprograms terminally differentiating cells to undergo unscheduled DNA synthesis. Shown are organotypic raft cultures of HPV16^WT-harboring NIKS cells (A), HPV16 E5^XCM−-harboring NIKS cells (B), and untransfected NIKS cells (C). BrdU was added to culture media 8 h prior to harvest. BrdU incorporation was detected by immunohistochemical staining using an antibody to BrdU. Shown is BrdU-specific immunohistochemical staining (brown nuclei) with hematoxylin counterstain (blue nuclei). BrdU was detected only in the basal compartment of untransfected NIKS cells (C). In contrast, BrdU was detected in both the basal and suprabasal compartment of HPV16^WT- and HPV16^E5XCM−-harboring NIKS (A and B). However, the graph (D) demonstrates that there is a quantitative reduction (approximately twofold) in the percentage of BrdU-positive cells in the supraparabasal compartment of HPV16^E5XCM− mutant-harboring rafts compared with that of HPV16^WT rafts (D). To obtain the data graphed, BrdU-positive cells from 7 populations of untransfected NIKS cells, 8 populations of independently derived populations of NIKS cells harboring HPV16^WT genomes, and 10 independently derived populations of NIKS cells harboring HPV16 E5^XCM− genomes were counted from four independent experiments (10 fields per slide at a magnification of ×40) as described in Materials and Methods.