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

Braeuning A, Vetter S - Biosci. Rep. (2012)

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Tübingen, Tübingen, Germany. albert.braeuning@uni-tuebingen.de

ABSTRACT
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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Improvement of the ability of a Renilla luciferasereaction buffer to quench firefly luciferase activity(A) Remnant activity of firefly luciferase inRenilla buffer is further reduced by addition of20 μM NFκBAI4. Values are given as percentage ofcontrol (i.e. lysate with firefly buffer prior to the addition ofRenilla buffer) (B)Renilla luciferase activity is not altered by thepresence of NFκBAI4. Renilla luciferase activitywas assayed using Renilla buffer with or without20 μM NFκBAI4 in lysates from transientlytransfected Hepa1c1c7 cells. Means±S.D. (n=6)are given; *P<0.05.
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Figure 4: Improvement of the ability of a Renilla luciferasereaction buffer to quench firefly luciferase activity(A) Remnant activity of firefly luciferase inRenilla buffer is further reduced by addition of20 μM NFκBAI4. Values are given as percentage ofcontrol (i.e. lysate with firefly buffer prior to the addition ofRenilla buffer) (B)Renilla luciferase activity is not altered by thepresence of NFκBAI4. Renilla luciferase activitywas assayed using Renilla buffer with or without20 μM NFκBAI4 in lysates from transientlytransfected Hepa1c1c7 cells. Means±S.D. (n=6)are given; *P<0.05.

Mentions: An inhibition of firefly luciferase might be desired under certain conditions:for example, dual luciferase assays normally consist of an analysis of fireflyluciferase activity followed by the assessment of Renillaluciferase activity in the same cell lysate. This implies that the activity offirefly luciferase is effectively quenched before measuring luminescenceproduced by the Renilla enzyme, an issue that is solved by achange of buffer composition and pH in the reaction tube. Using our buffers,residual activity of firefly luciferase is ~0.02% of the initialactivity, as determined by measuring lysates containing only firefly but notRenilla luciferase before and after addition of theRenilla buffer (Figure4A). The addition of 20 μM NFκBAI4 to theRenilla buffer further improved its ability to quench thefirefly signal (Figure 4A), but did notinfluence Renilla luciferase activity, as determined by theaddition of 20 μM NFκBAI4 to the Renillaluciferase reaction buffer in a classic dual luciferase assay with transientlytransfected Hepa1c1c7 cells (Figure 4B).Thus, in principle, dual luciferase systems might be improved by addition of afirefly luciferase inhibitor to the Renilla luciferase reactionbuffer.


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

Braeuning A, Vetter S - Biosci. Rep. (2012)

Improvement of the ability of a Renilla luciferasereaction buffer to quench firefly luciferase activity(A) Remnant activity of firefly luciferase inRenilla buffer is further reduced by addition of20 μM NFκBAI4. Values are given as percentage ofcontrol (i.e. lysate with firefly buffer prior to the addition ofRenilla buffer) (B)Renilla luciferase activity is not altered by thepresence of NFκBAI4. Renilla luciferase activitywas assayed using Renilla buffer with or without20 μM NFκBAI4 in lysates from transientlytransfected Hepa1c1c7 cells. Means±S.D. (n=6)are given; *P<0.05.
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Related In: Results  -  Collection

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Figure 4: Improvement of the ability of a Renilla luciferasereaction buffer to quench firefly luciferase activity(A) Remnant activity of firefly luciferase inRenilla buffer is further reduced by addition of20 μM NFκBAI4. Values are given as percentage ofcontrol (i.e. lysate with firefly buffer prior to the addition ofRenilla buffer) (B)Renilla luciferase activity is not altered by thepresence of NFκBAI4. Renilla luciferase activitywas assayed using Renilla buffer with or without20 μM NFκBAI4 in lysates from transientlytransfected Hepa1c1c7 cells. Means±S.D. (n=6)are given; *P<0.05.
Mentions: An inhibition of firefly luciferase might be desired under certain conditions:for example, dual luciferase assays normally consist of an analysis of fireflyluciferase activity followed by the assessment of Renillaluciferase activity in the same cell lysate. This implies that the activity offirefly luciferase is effectively quenched before measuring luminescenceproduced by the Renilla enzyme, an issue that is solved by achange of buffer composition and pH in the reaction tube. Using our buffers,residual activity of firefly luciferase is ~0.02% of the initialactivity, as determined by measuring lysates containing only firefly but notRenilla luciferase before and after addition of theRenilla buffer (Figure4A). The addition of 20 μM NFκBAI4 to theRenilla buffer further improved its ability to quench thefirefly signal (Figure 4A), but did notinfluence Renilla luciferase activity, as determined by theaddition of 20 μM NFκBAI4 to the Renillaluciferase reaction buffer in a classic dual luciferase assay with transientlytransfected Hepa1c1c7 cells (Figure 4B).Thus, in principle, dual luciferase systems might be improved by addition of afirefly luciferase inhibitor to the Renilla luciferase reactionbuffer.

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Tübingen, Tübingen, Germany. albert.braeuning@uni-tuebingen.de

ABSTRACT
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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