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. 2013 Dec 3;289(3):1866–1875. doi: 10.1074/jbc.M113.505578

FIGURE 1.

FIGURE 1.

Increased expression of AhR and CYP1A1 by LPS in human DC and thymus of B6 mice. A, LPS induces AhR and CYP1A1 in human DC. Human monocyte-derived DC were generated as described under “Experimental Procedures.” DC were treated with 10 nm TCDD for 7 days or 0.05 μg/ml LPS for 24 h. TCDD-treated DC were co-treated with LPS for 24 h. B6 wild type (B) and Ahr−/− (C) mice were treated with 15 μg/kg TCDD for 24 h or with 0.5 mg/kg LPS for 6 h. For co-treatment, mice were treated with 15 μg/kg TCDD for 18 h and then treated with 0.5 mg/kg LPS for 6 h. D, time course study of AhR, ARNT, and CYP1A1 mRNA induction in U937-derived DC. Cells were treated for 1 to 48 h with 0.1 μg/ml LPS or 1 μl/ml water (Control). E, Western blot analysis of AhR, ARNT, and CYP1A1 protein level in human DC. 25 μg of whole cell protein was loaded per lane. AhR and CYP1A1 protein levels were quantitated and normalized to actin. Values represent the mean ± S.D. of three independent experiments. An asterisk indicates significantly different from control cells (p < 0.05). Double asterisks indicate significantly higher than only TCDD-treated cells (p < 0.05). F, LPS-induced AhR binding to a DRE consensus element of the Cyp1a1 promoter. Nuclear extracts from untreated control (lane 1) and LPS-treated (lanes 3 and 5) U937-derived DC were used for EMSA. Cells were treated with 1 nm TCDD for 1 h or with 0.1 μg/ml LPS for 6 h. A possible enhancement of TCDD-induced AhR-binding activity as shown in lane 2 was tested by treatment of cells with LPS (0.1 μg/ml) for 6 h followed by treatment with TCDD (1 nm) for 1 h (lane 4). EMSA was performed using double-stranded, 32P-labeled oligonucleotide containing the DRE binding sequence of the rat Cyp1a1 promoter. To confirm specificity, a 100-fold excess of unlabeled DRE oligonucleotide (lane 5) was added. G, the specific binding of AhR and ARNT was identified by EMSA supershift analyses using AhR- and ARNT-specific antibodies (lanes 5 and 6). For EMSA, one representative experiment of three independently performed experiments is shown. Ab., antibody; Comp., competition; Ctrl, control; Treat., treatment.