FIG. 4.

Delayed accumulation of abundant p27kip1, cyclin E, and p21cip1 proteins in HPV-18 URR-E7-transduced, differentiated keratinocytes that have reentered S phase prevents further rounds of S phase. The cultures were those described in Fig. 1A (top), except that BrdU was not assayed in these experiments. Sections were immunostained for the protein of interest (in red) and then autoradiographed to reveal ³H-TdR incorporation. (A) ³H-TdR labeling scheme and the percentage of cells positive for both ³H-TdR and cyclin E or p27kip1 among ³H-TdR-positive cells in the differentiated strata. (B) Cyclin E immunostaining in control and E7-transduced raft cultures. Immunostaining of p21cip1 (C) and p27kip1 (D) in control and E7-transduced cultures. The insets in panels B and D are enlarged images of selected (boxed) binucleated, doubly positive cells. The inset in panel C is an enlarged view of the selected (boxed), doubly positive cell with an enlarged nucleus. (E) Images of control and E7-transduced raft cultures that had been exposed to BrdU continuously for 96 h. BrdU incorporation was revealed by FITC-conjugated anti-BrdU (green), and nuclei were stained by DAPI (blue). Arrows in panels B through E point to the basal stratum.