Figure 3. VDRM2 is not metabolized in VCaP cells.

(A) Diagram of the experimental procedure. VCaP cells were treated with vehicle (EtOH), 30 nM, 100 nM, or 300 nM 1,25D(OH)₂D₃ (1,25D), or 300 nM VDRM2 for 96 or 24 hours (Conditioned medium). Plates with no cells were incubated with the same concentrations of agonists for 96 hours (Mock) and fresh medium was prepared with the same concentrations of agonists at the end of the 96 hour incubation (Control). 293T cells were transfected with a VDR-RXR two-hybrid luciferase reporter as described in methods. A total of 100 μl of conditioned medium from VCaP cells in Figure 2 as well as media from the mock and control conditions were added to the transfected 293T cells in 1.5 ml of medium for 24 hours and then harvested. The cells were lysed and luciferase activity was measured and normalized to β-galactosidase activity. (B) Media assayed from VCaP cells treated with vehicle (EtOH), sub-maximum concentrations of 1,25D(OH)₂D₃ (30 nM), or 300 nM VDRM2 from Figure 2A, 2B. (C) Media assayed from VCaP cells treated with vehicle (EtOH) or higher concentrations of 1,25D(OH)₂D₃ (100 nM or 300 nM) from Figure 2C, 2D.**p < 0.01, ***p < 0.001, relative to respective 24 hour time point. n = 3, representative graph, mean ± SEM.