FIG. 6.

Analysis of DNA synthesis and viral DNA amplification in organotypic raft cultures of NIKS cells alone or NIKS cells harboring the wild-type or E4 mutant HPV16 genomes. To identify cells supporting DNA synthesis, histological sections (A to E and K to O) from raft cultures that were incubated in medium containing BrdU for 8 h prior to harvest were immunohistochemically stained with anti-BrdU antibody, and the nuclei were counterstained with hematoxylin (blue). In cells that had incorporated BrdU, nuclei were stained brown. To detect cells in which the HPV16 genome is amplified, serial histological sections (F to J and P to T) were subjected to HPV16-specific FISH. Cells containing amplified HPV16 DNA were detected with FITC-conjugated anti-DIG antibody (Roche; green) and counterstained with DAPI (Vector) to indicate nuclei (blue). The arrows in panel G indicate punctate, small dots that are found only in rafts harboring the 16st10 genome, which show a different pattern of signal from rafts harboring the wild-type or other E4 mutant HPV16 genomes. Raft cultures of NIKS cells alone (A and P) and NIKS cells harboring the wild-type HPV16 genome (A and F), E4 mutant HPV16 genome 16st10 (B and G), 16st15 (C and H), 16st20 (D and I), 16st32 (E and J), 16st67(L and Q), 16st84 (M and R), PSS/WT (N and S), and PSS/20 (O and T) are shown.