Real-Time Monitoring of Nuclear Factor κB Activity in Cultured Cells and in Animal Models

Nuclear factor κB (NF-κB) is a transcription factor that plays a major role in many human disorders, including immune diseases and cancer. We designed a reporter system based on NF-κB responsive promoter elements driving expression of the secreted Gaussia princeps luciferase (Gluc). We show that this bioluminescent reporter is a highly sensitive tool for noninvasive monitoring of the kinetics of NF-κB activation and inhibition over time, both in conditioned medium of cultured cells and in the blood and urine of animals. NF-κB activation was successfully monitored in real time in endothelial cells in response to tumor angiogenic signaling, as well as in monocytes in response to inflammation. Further, we demonstrated dual blood monitoring of both NF-κB activation during tumor development as correlated to tumor formation using the NF-κB Gluc reporter, as well as the secreted alkaline phosphatase reporter. This NF-κB reporter system provides a powerful tool for monitoring NF-κB activity in real time in vitro and in vivo.

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Aurora B Overexpression Causes Aneuploidy and p21Cip1Repression during Tumor Development

Molecular and Cellular Biology ◽

10.1128/mcb.01286-14 ◽

2015 ◽

Vol 35 (20) ◽

pp. 3566-3578 ◽

Cited By ~ 47

Author(s):

Alejandra González-Loyola ◽

Gonzalo Fernández-Miranda ◽

Marianna Trakala ◽

David Partida ◽

Kumiko Samejima ◽

...

Keyword(s):

Chromosome Segregation ◽

Cultured Cells ◽

Tumor Development ◽

Inverse Correlation ◽

Aurora Kinase ◽

Tumor Formation ◽

Aurora B ◽

Aurora Kinase B

Aurora kinase B, one of the three members of the mammalian Aurora kinase family, is the catalytic component of the chromosomal passenger complex, an essential regulator of chromosome segregation in mitosis. Aurora B is overexpressed in human tumors although whether this kinase may function as an oncogenein vivois not established. Here, we report a new mouse model in which expression of the endogenousAurkblocus can be inducedin vitroandin vivo. Overexpression of Aurora B in cultured cells induces defective chromosome segregation and aneuploidy. Long-term overexpression of Aurora Bin vivoresults in aneuploidy and the development of multiple spontaneous tumors in adult mice, including a high incidence of lymphomas. Overexpression of Aurora B also results in a reduced DNA damage response and decreased levels of the p53 target p21Cip1in vitroandin vivo, in line with an inverse correlation between Aurora B and p21Cip1expression in human leukemias. Thus, overexpression of Aurora B may contribute to tumor formation not only by inducing chromosomal instability but also by suppressing the function of the cell cycle inhibitor p21Cip1.

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Periprosthetic Osteolysis: Mechanisms, Prevention and Treatment

Journal of Clinical Medicine ◽

10.3390/jcm8122091 ◽

2019 ◽

Vol 8 (12) ◽

pp. 2091 ◽

Cited By ~ 14

Author(s):

Stuart B. Goodman ◽

Jiri Gallo

Keyword(s):

Nuclear Factor Κb ◽

Nuclear Factor ◽

Activator Protein 1 ◽

Periprosthetic Osteolysis ◽

Bone Implant ◽

Joint Replacements ◽

In Vivo Experiments ◽

Mechanical Factors

Clinical studies, as well as in vitro and in vivo experiments have demonstrated that byproducts from joint replacements induce an inflammatory reaction that can result in periprosthetic osteolysis (PPOL) and aseptic loosening (AL). Particle-stimulated macrophages and other cells release cytokines, chemokines, and other pro-inflammatory substances that perpetuate chronic inflammation, induce osteoclastic bone resorption and suppress bone formation. Differentiation, maturation, activation, and survival of osteoclasts at the bone–implant interface are under the control of the receptor activator of nuclear factor kappa-Β ligand (RANKL)-dependent pathways, and the transcription factors like nuclear factor κB (NF-κB) and activator protein-1 (AP-1). Mechanical factors such as prosthetic micromotion and oscillations in fluid pressures also contribute to PPOL. The treatment for progressive PPOL is only surgical. In order to mitigate ongoing loss of host bone, a number of non-operative approaches have been proposed. However, except for the use of bisphosphonates in selected cases, none are evidence based. To date, the most successful and effective approach to preventing PPOL is usage of wear-resistant bearing couples in combination with advanced implant designs, reducing the load of metallic and polymer particles. These innovations have significantly decreased the revision rate due to AL and PPOL in the last decade.

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The nuclear factor κB inhibitor (E)-2-fluoro-4′-methoxystilbene inhibits firefly luciferase

Bioscience Reports ◽

10.1042/bsr20120043 ◽

2012 ◽

Vol 32 (6) ◽

pp. 531-537 ◽

Cited By ~ 16

Author(s):

Albert Braeuning ◽

Silvia Vetter

Keyword(s):

Photon Emission ◽

Gene Promoter ◽

Firefly Luciferase ◽

Nuclear Factor Κb ◽

Signalling Pathway ◽

Luciferase Reporter ◽

Nuclear Factor ◽

Reporter System ◽

Reporter Assays

Photinus pyralis (firefly) luciferase is widely used as a reporter system to monitor alterations in gene promoter and/or signalling pathway activities in vitro. The enzyme catalyses the formation of oxyluciferin from D-luciferin in an ATP-consuming reaction involving photon emission. The purpose of the present study was to characterize the luciferase-inhibiting potential of (E)-2-fluoro-4′-methoxystilbene, which is known as a potent inhibitor of the NF-κB (nuclear factor κB) signalling pathway that is used to modulate the NF-κB signalling pathway in vitro. Results show that (E)-2-fluoro-4′-methoxystilbene effectively inhibits firefly luciferase activity in cell lysates and living cells in a non-competitive manner with respect to the luciferase substrates D-luciferin and ATP. By contrast, the compound has no effect on Renilla and Gaussia luciferases. The mechanism of firefly luciferase inhibition by (E)-2-fluoro-4′-methoxystilbene, as well as its potency is comparable to its structure analogue resveratrol. The in vitro use of trans-stilbenes such as (E)-2-fluoro-4′-methoxystilbene or resveratrol compromises firefly luciferase reporter assays as well as ATP/luciferase-based cell viability assays.

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In Vitro and in Vivo Nuclear Factor-κB Inhibitory Effects of the Cell-Penetrating Penetratin Peptide

Molecular Pharmacology ◽

10.1124/mol.105.019653 ◽

2006 ◽

Vol 69 (6) ◽

pp. 2027-2036 ◽

Cited By ~ 17

Author(s):

Tamás Letoha ◽

Erzsébet Kusz ◽

Gábor Pápai ◽

Annamária Szabolcs ◽

József Kaszaki ◽

...

Keyword(s):

Nuclear Factor Κb ◽

Nuclear Factor ◽

Inhibitory Effects ◽

Cell Penetrating

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Fucoidans from Cucumaria frondosa ameliorates renal interstitial fibrosis via inhibition of PI3K/Akt/NF-κB signaling pathway

Food & Function ◽

10.1039/d1fo03067a ◽

2022 ◽

Author(s):

Zhuo-yue Song ◽

Mengru Zhu ◽

Jun Wu ◽

Tian Yu ◽

Yao Chen ◽

...

Keyword(s):

Protein Kinase ◽

Signaling Pathway ◽

Interstitial Fibrosis ◽

Protein Kinase B ◽

Nuclear Factor Κb ◽

Nuclear Factor ◽

Renal Interstitial Fibrosis ◽

Cucumaria Frondosa

The effects of Cucumaria frondosa polysaccharides (CFP) on renal interstitial fibrosis via regulating phosphatidylinositol-3-hydroxykinase/protein kinase-B/Nuclear factor-κB (PI3K/AKT/NF-κB) signaling pathway were investigated in vivo and in vitro in this research. A...

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