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. Author manuscript; available in PMC: 2008 May 9.

Published in final edited form as: Mol Endocrinol. 2007 Sep 6;21(12):2941–2955. doi: 10.1210/me.2007-0211

Fig. 2 — RelB and AhR mediate FSK- and TCDD-induced IL-8 activation. A, The AP-1, the Oct-1, and the NF-κB sites of the 5′-flanking region of the IL-8 gene are located 126 bp, 94 bp, and 80 bp, respectively, upstream of the start site of transcription in the IL-8 gene. U937 cells were transiently transfected with deletion constructs corresponding to the first 272, 137, 98, or 50 bp of the 5′-flanking region of the IL-8 gene and treated with 10 nM TCDD or 10 μM FSK for 24 h. Relative luciferase units are given as mean values of triplicates as a result of three independent experiments. *, significantly different from control (p<0.001). B, Supershift analyses with p50-, RelA-, RelB-, AhR-, and ARNT-specific antibodies were performed with a ³²P-end-labeled oligonucleotide (5′-AGATGAGGGTGCATAAGTTC-3′) containing the RelBAhRE site of the IL-8 gene with nuclear extracts of untreated and TCDD-stimulated cells treated for 90 min. A 100-fold molar excess of unlabeled RelBAhRE was added. C, Densitometric evaluation of band intensities of the RelB/AhR complexes. Results of three independent experiments are shown as mean values ± S.D. *, significantly different from control cells (p<0.001). D, Nucleotide sequence of the wild-type (wt) -50 bp IL-8 construct corresponding to the 5′-flanking region of the first -120 bp upstream of the start site. The TATA box is in italic type, the AP-1, Oct-1, and NF-κB sites are underlined, the RelBAhRE site is shown in boldface letters. A one point mutation (M1) or two point mutations (M2) were introduced in the RelBAhRE site of the -50 bp construct. E, DNA binding of nuclear proteins from U937 macrophages to the RelBAhRE probe of the IL-8 promoter or RelBAhRE with two different point mutations M1 and M2. U937 macrophages were treated with 10 nM TCDD (T), 2 μg/ml LPS (L), 10 μM FSK (F), or received 0.1% Me₂SO as vehicle control (C), and nuclear proteins were extracted at 90 min. A 100-fold molar excess of unlabeled wildtype oligonucleotides was added. F, U937 cells were transiently transfected with -50 wt IL-8 construct and the mutation constructs M1 or M2. After transfection, cells were treated with 10 nM TCDD or 10 μM FSK for 24 h. Relative luciferase activity units are given as mean values of triplicates as a result of three independent experiments. *, significantly different from control (p<0.005); **, significantly higher than only -50 wt transfected cells treated with TCDD or FSK (p<0.005). G, DNA binding of nuclear proteins from U937 macrophages to oligos containing a point mutation M1 of the RelBAhRE probe of the IL-8 promoter. U937 macrophages were treated with 10 μM FSK (F), 10 nM TCDD (T), or received 0.1% Me₂SO as vehicle control (C), and nuclear proteins were extracted at 90 min. Supershift analyses with p50-, RelA-, ARNT-, RelB-, or AhR-specific antibodies were performed to identify proteins binding to the mutated M1 RelBAhRE sequence of IL-8. A 100-fold molar excess of unlabeled oligonucleotide was added.

Fig. 2 — RelB and AhR mediate FSK- and TCDD-induced IL-8 activation. A, The AP-1, the Oct-1, and the NF-κB sites of the 5′-flanking region of the IL-8 gene are located 126 bp, 94 bp, and 80 bp, respectively, upstream of the start site of transcription in the IL-8 gene. U937 cells were transiently transfected with deletion constructs corresponding to the first 272, 137, 98, or 50 bp of the 5′-flanking region of the IL-8 gene and treated with 10 nM TCDD or 10 μM FSK for 24 h. Relative luciferase units are given as mean values of triplicates as a result of three independent experiments. *, significantly different from control (p<0.001). B, Supershift analyses with p50-, RelA-, RelB-, AhR-, and ARNT-specific antibodies were performed with a ³²P-end-labeled oligonucleotide (5′-AGATGAGGGTGCATAAGTTC-3′) containing the RelBAhRE site of the IL-8 gene with nuclear extracts of untreated and TCDD-stimulated cells treated for 90 min. A 100-fold molar excess of unlabeled RelBAhRE was added. C, Densitometric evaluation of band intensities of the RelB/AhR complexes. Results of three independent experiments are shown as mean values ± S.D. *, significantly different from control cells (p<0.001). D, Nucleotide sequence of the wild-type (wt) -50 bp IL-8 construct corresponding to the 5′-flanking region of the first -120 bp upstream of the start site. The TATA box is in italic type, the AP-1, Oct-1, and NF-κB sites are underlined, the RelBAhRE site is shown in boldface letters. A one point mutation (M1) or two point mutations (M2) were introduced in the RelBAhRE site of the -50 bp construct. E, DNA binding of nuclear proteins from U937 macrophages to the RelBAhRE probe of the IL-8 promoter or RelBAhRE with two different point mutations M1 and M2. U937 macrophages were treated with 10 nM TCDD (T), 2 μg/ml LPS (L), 10 μM FSK (F), or received 0.1% Me₂SO as vehicle control (C), and nuclear proteins were extracted at 90 min. A 100-fold molar excess of unlabeled wildtype oligonucleotides was added. F, U937 cells were transiently transfected with -50 wt IL-8 construct and the mutation constructs M1 or M2. After transfection, cells were treated with 10 nM TCDD or 10 μM FSK for 24 h. Relative luciferase activity units are given as mean values of triplicates as a result of three independent experiments. *, significantly different from control (p<0.005); **, significantly higher than only -50 wt transfected cells treated with TCDD or FSK (p<0.005). G, DNA binding of nuclear proteins from U937 macrophages to oligos containing a point mutation M1 of the RelBAhRE probe of the IL-8 promoter. U937 macrophages were treated with 10 μM FSK (F), 10 nM TCDD (T), or received 0.1% Me₂SO as vehicle control (C), and nuclear proteins were extracted at 90 min. Supershift analyses with p50-, RelA-, ARNT-, RelB-, or AhR-specific antibodies were performed to identify proteins binding to the mutated M1 RelBAhRE sequence of IL-8. A 100-fold molar excess of unlabeled oligonucleotide was added.