Blockade of MEK1/MEK2 activity abolishes 1,25D-induction of AP-1 and p21, and prevents hormone anti-proliferative effects. Native, non-transfected (Non-T) and control vector transfected (V-T) SaOS-2 cells, co-transfected with AP-1 (x7) enhancer (A) or p21 promoter (B) luciferase constructs, were cultured for 3 days in the presence of 10 nM 1,25D, with (filled bars) or without (open bars) specific MEK1/MEK2 blocker U0126 (25 μM). Relative light units (RLU) were measured as a function of AP-1 enhancer and p21 promoter activities. Results show that inhibition of MEK1/MEK2 signaling prevented AP-1 and p21 upregulation, confirming that p21 gene transactivation occurs downstream 1,25D-induced MAPK activity. *, p<0.001, n=4. C: Measurements for cell densities obtained for native (non-transfected) SaOS-2 cell cultures treated with 10 nM 1,25D in the presence or absence of U0126 (25 μM) for 3 days. *, p<0.01, n=3.