1. Arterioscler Thromb Vasc Biol
Dysregulation of Monocytic Nuclear Factor-κB by Oxidized Low-Density Lipoprotein
by Korbinian Brand, Tamara Eisele, Ursula Kreusel, Michael Page, Sharon Page, Monika Haas, Astrid Gerling, Christian Kaltschmidt, Franz-Josef Neumann, Nigel Mackman, Patrick A. Baeuerle, Autar K. Walli, and Dieter Neumeier
Arterioscler Thromb Vasc Biol
Volume 17(10):
October 1, 1997
Copyright © American Heart Association, Inc. All rights reserved.

2. Activation of NF-κB by oxLDL in monocytic cells.
Activation of NF-κB by oxLDL in monocytic cells. A, THP-1 cells were incubated for 1 hour in the absence of lipoproteins or with 80 μg/mL of oxLDL or LDL, respectively. Activation of NF-κB was determined by EMSA; bracket indicates the position of NF-κB bands. Binding of nuclear proteins to an oligonucleotide containing the Sp-1 consensus sequence was also examined in the same nuclear extracts (double arrow). B, Nuclear appearance of activated NF-κB in the presence of oxLDL. Activated NF-κB was detected by immunocytochemistry using the monoclonal antibody α-p65MAb (dilution 1:500) and BDHC staining, giving a blue-black color in the original micrograph and a granular staining pattern. The activated NF-κB appears to be present mostly in the nucleus of oxLDL-treated cells. Magnification ×240. Co indicates control.
Korbinian Brand et al. Arterioscler Thromb Vasc Biol. 1997;17:
Copyright © American Heart Association, Inc. All rights reserved.

3. oxLDL induces NF-κB activation and associated target gene expression in adherent monocytes.
oxLDL induces NF-κB activation and associated target gene expression in adherent monocytes. Human adherent monocytes were incubated for 4 hours in the absence of lipoproteins or with 80 μg/mL of LDL or oxLDL, respectively. Activated NF-κB was measured by EMSA and is indicated by the bracket (A). In the same set of experiments the production of IL-8 in the supernatant was detected by immunoassay (B). Co indicates control.
Korbinian Brand et al. Arterioscler Thromb Vasc Biol. 1997;17:
Copyright © American Heart Association, Inc. All rights reserved.

4. IL-8 promoter-dependent transcription is induced by oxLDL.
IL-8 promoter-dependent transcription is induced by oxLDL. THP-1 cells were transiently cotransfected with a luciferase reporter gene construct containing 420 bp of the 5′-region of the IL-8 gene (pGL2 IL-8) or lacking this region (pGL2 Basic) and pRLtk Renilla luciferase control plasmid. Transfected cells were incubated with LDL or oxLDL (80 μg/mL) for 5 hours. Data are expressed as firefly luciferase RLU divided by Renilla-RLU.
Korbinian Brand et al. Arterioscler Thromb Vasc Biol. 1997;17:
Copyright © American Heart Association, Inc. All rights reserved.

5. Composition of oxLDL-induced complexes.
Composition of oxLDL-induced complexes. Nuclear extracts harvested from (A) oxLDL-treated THP-1 cells (1-hour incubation) and (B) adherent monocytes (4-hour incubation) were analyzed by supershift analysis. For this purpose, nuclear extracts were preincubated with polyclonal antibodies directed against p50, p65, and c-Rel or with p65 and c-Rel together. Brackets indicate position of the NF-κB bands. Arrows mark the positions of the bands shifted by preincubation with anti-p65 or anti-p50. No Ab indicates that no antibody was added for preincubation.
Korbinian Brand et al. Arterioscler Thromb Vasc Biol. 1997;17:
Copyright © American Heart Association, Inc. All rights reserved.

6. Effects of the antioxidant PDTC and the proteasome inhibitor PSI
Effects of the antioxidant PDTC and the proteasome inhibitor PSI. THP-1 cells were incubated in plain medium or with 80 μg/mL of oxLDL in the absence or presence of different concentrations of PDTC for 6 hours (A) or pretreated with increasing doses of PSI for 1 hour and then stimulated with lipoproteins for 1 hour (B).
Effects of the antioxidant PDTC and the proteasome inhibitor PSI. THP-1 cells were incubated in plain medium or with 80 μg/mL of oxLDL in the absence or presence of different concentrations of PDTC for 6 hours (A) or pretreated with increasing doses of PSI for 1 hour and then stimulated with lipoproteins for 1 hour (B). NF-κB was examined by EMSA; brackets indicate activated NF-κB. Cytosolic IκB-α was detected by Western blot analysis and is marked by arrows. Note that in most of the experiments the slower-migrating phosphorylated form of IκB-α could be detected as marked by the upper arrow of the doublet.
Korbinian Brand et al. Arterioscler Thromb Vasc Biol. 1997;17:
Copyright © American Heart Association, Inc. All rights reserved.

7. Time course of oxLDL-induced NF-κB activation and IκB-α depletion.
Time course of oxLDL-induced NF-κB activation and IκB-α depletion. THP-1 cells were incubated over 24 hours with 80 μg/mL of oxLDL. NF-κB activation was determined by EMSA and cytosolic IκB-α was detected by Western blot analysis. Data were analyzed by densitometry and are combined on a histogram. NF-κB activation is depicted as arbitrary units normalized against Sp-1 binding examined in the same extracts. For the IκB-α data, the level of this inhibitor in the untreated control (0 hours) was defined as the 100% value.
Korbinian Brand et al. Arterioscler Thromb Vasc Biol. 1997;17:
Copyright © American Heart Association, Inc. All rights reserved.

8. Long-term exposure to oxLDL inhibits LPS-induced NF-κB activation and target gene expression.
Long-term exposure to oxLDL inhibits LPS-induced NF-κB activation and target gene expression. A, Inhibition of LPS-induced NF-κB activation by oxLDL. THP-1 cells were preincubated in medium alone, 80 μg/mL LDL, or oxLDL for 24 hours followed by a 1-hour stimulation with 1 μg/mL LPS. Activated NF-κB, examined by EMSA, is indicated by a bracket. Binding of nuclear proteins to an oligonucleotide containing the Sp-1 consensus sequence was also examined in the same nuclear extracts (double arrow). B, Inhibition of LPS-induced TNF-α and IL-1β mRNA expression by oxLDL. The cells were incubated as mentioned in A, with the only exception that total RNA was harvested for Northern blot analysis after a 3-hour stimulation with LPS. To account for variability in sample loading, the blot was rehybridized with a GAPDH cDNA probe.
Korbinian Brand et al. Arterioscler Thromb Vasc Biol. 1997;17:
Copyright © American Heart Association, Inc. All rights reserved.

9. Long-term exposure to oxLDL prevents the efficient activation-induced depletion of IκB-α.
Long-term exposure to oxLDL prevents the efficient activation-induced depletion of IκB-α. A, THP-1 cells were preincubated in medium alone or 80 μg/mL oxLDL for 24 hours followed by a 1-hour stimulation with 1 μg/mL LPS. Cytosolic IκB-α was determined by Western blot analysis and is indicated by the arrows. The slower migrating phosphorylated form of IκB-α that could be detected in most of the experiments is marked by the upper arrow of the doublet. The positions of the molecular weight (MW) marker proteins are indicated. B, Cells were preincubated in the absence of lipoproteins or with 80 μg/mL LDL or oxLDL for 24 hours and then stimulated with 1 μg/mL LPS for 1 hour. Cytosolic IκB-α was measured as described in A.
Korbinian Brand et al. Arterioscler Thromb Vasc Biol. 1997;17:
Copyright © American Heart Association, Inc. All rights reserved.