FIG. 2.

Functional activation of HPV-16 E7ER in PHK raft cultures by E2. The raft cultures were left untreated (0 h) or treated with E2 for the specified durations prior to harvest on day 9. BrdU was added 12 h (A and B) or 6 h (C) prior to harvest. Immunohistochemistry to detect BrdU incorporation (in red) (A and B) was performed on 4-μm sections to reveal S-phase cells. (A) Raft cultures expressing ER after induction with 1 μM E2 for 0 h, 24 h, 36 h, or 48 h. (B) Raft cultures expressing 16E7ER in the absence of E2 or in the presence of 1 μM E2 for 24 h, 36 h, or 48 h (upper images) or after induction for 24 h with 0.1 μM, 1.0 μM, 5.0 μM, or 10 μM E2 (lower images). (C) Double immunofluorescence revealing time-dependent induction of p21cip1 protein stabilization (red) or S-phase reentry, as indicated by BrdU incorporation (green), in separate populations of differentiated cells in the presence of 5 μM of E2 for 0 h, 6 h, 12 h, or 24 h. The arrows point to the basal stratum.