FIGURE 5.

PMA induces p21^Cip1 mRNA levels and activates a human p21^Cip1 luciferase reporter. A, LNCaP cells were co-transfected with a p21^Cip1 firefly luciferase reporter and pTK-Renilla. Forty-eight h later, cells were treated with either 100 nm PMA or vehicle for 1 h, in the presence of Y-27632 (10 μm) or cytochalasin B (2 μm). Luciferase activity was determined at 9 h after PMA treatment and normalized to Renilla luciferase activity. B, LNCaP cells were co-transfected with p21^Cip1 firefly luciferase reporters with different deletions in the promoter region and pTK-Renilla. Forty-eight h later, cells were treated with either 100 nm PMA or vehicle for 1 h. Luciferase activity was determined at 9 h after PMA treatment and normalized to Renilla luciferase activity. Results are expressed as -fold change relative to vehicle-treated cells and presented as mean ± S.D. (n = 3). Similar results were observed in three experiments. C, LNCaP cells subject to Sp1 RNAi depletion were treated with 100 nm PMA for 1 h, and p21^Cip1 induction was determined by Western blot. Left, representative Sp1 depletion. Upper right, representative p21^Cip1 induction. Lower right, densitometric analysis of three independent experiments, expressed as mean ± S.D. *, p < 0.05 versus control, 8 h.