The roles of the Vdr and Stat1 for the 1α,25(OH)₂D₃-mediated inhibitory effects. (A) Real-time Vdr RT-PCR. BMDMs were incubated with IFN-γ or 1α,25(OH)₂D₃ or both for 48 hours, and total RNA was isolated and reverse transcribed. Vdr-specific primers were used for amplification and mRNA expression was normalized as described (= relative mRNA expression). Synergistic induction of Vdr mRNA by IFN-γ and 1α,25(OH)₂D₃ was confirmed in 3 additional experiments. (B) Time kinetics of Vdr mRNA induction. Vdr transcript levels were determined as described in panel A using real-time RT-PCR. (C) Intracellular localization of Vdr protein. BMDMs were cultured as described in panel A, fixed, and stained with an anti-Vdr antibody (green) and analyzed by confocal microscopy. (D) The inhibition of O₂^{- ·} production by 1α,25(OH)₂D₃ is dependent on a functional Vdr. BMDMs isolated from WT and Vdr-KO mice were cultured with IFN-γ and 1α,25(OH)₂D₃ and intracellular production of O₂^{- ·} was analyzed. (E) The 1α,25(OH)₂D₃-mediated inhibition of listericidal activity depends on the Vdr. BMDMs from WT and Vdr-KO mice were cultured with IFN-γ and 1α,25(OH)₂D₃ and infected with opsonized L monocytogenes for 3 hours as described in Figure 1. Data are presented as the mean ± SEM calculated from triplicate wells (plating was carried out in duplicate). (F) Real-time RT-PCR of Cybb. BMDMs (WT and Vdr-KO) were cultured as described in panel A. Cybb-specific primers were used for amplification and mRNA expression was normalized as described. The experiment was repeated 3 times with similar results. (G) Quantification of activated Stat1 after 1α,25(OH)₂D₃ and IFN-γ treatment. BMDMs were cultured as described in panel A and the amount of tyrosine 701-phosphorylated Stat1 protein was determined. ^*P < 0.05; Wilcoxon-signed rank test. Shown is a representative of at least 3 independent experiments (C-G).