Inducible DNase I hypersensitivity within the IL-12(p35) promoter. (A) Induction of IL-12(p35) mRNA in THP-1 cells. THP-1 cells were left untreated or were treated with LPS (1 μg/mL) and IFN-γ (100 U/mL) for 6 hours. Total RNA was reverse transcribed followed by real-time PCR with either IL-12(p35) or β-actin primers, using a one-step procedure. IL-12(p35) mRNA levels were normalized using β-actin mRNA levels and compared with unstimulated conditions. (B) Schematic representation of the probe used to map the nuclease-hypersensitive sites within the p35 promoter by the indirect end-labeling technique. Purified nuclei were treated with limiting concentrations of different nucleases (DNase I, MNase, or restriction enzymes). After isolation of genomic DNA, we performed overnight EcoRI digestion and analyzed the double-restriction products by Southern blotting using the indirect end-labeling technique. The probe spans nucleotides –1615 to –894 and is abutting the EcoRI restriction site. In vitro cleavage of genomic DNA by EcoRI yields an approximate 6-kb fragment. (C) In these experiments, THP-1 cells were either left untreated or treated with LPS (1 μg/mL) and IFN-γ (100 U/mL) for 4 to 6 hours. Nuclei were then purified and digested with increasing concentrations of DNase I (ranging from 0 to 50 U/mL). After purification of genomic DNA and in vitro digestion with EcoRI, DNA samples were analyzed by Southern blotting. Size markers are described in “Materials and methods.” The DNase I-hypersensitive site is indicated by a gray box. Increase in hypersensitivity on LPS/IFN-γ stimulation is indicated by a black extension of the gray box. (D) As a control, purified genomic DNA from THP-1 cells was digested in vitro with increasing concentrations of DNase I (ranging from 0.078 to 0.3125 U/mL). The results are representative of at least 3 independent experiments.