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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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Time-dependent firefly luciferase inhibition byNFκBAI4 in cell culture and analysis of the inhibitionmechanismLuciferase activity was assessed in lysates from various cell lines,derived from 70.4 or 55.1c mouse hepatoma cells, with stable expressionof firefly luciferase. Then 20 μM NFκBAI4 was addedto cell cultures and incubated for 1 h (A),24 h (B) or 48 h (C) prior tolysis and measurement. Luciferase signals were normalized to cellvitality, as determined by the Alamar Blue assay. Means±S.D.(n=3 and 4) are given;*P<0.05. For comparison see data forfirefly luciferase inhibition by resveratrol in Supplementary Figure S2at http://www.bioscirep.org/bsr/032/bsr0320531add.htm.(D) Dose-response analysis of firefly luciferaseactivity for the substrates D-luciferin and ATP in the presenceof different amounts of NFκBAI4. When varyingD-luciferin content of the reaction mixture, ATP was keptconstant at 500 μM; when varying ATP levels,D-luciferin was kept constant at 1000 μM. Solidlines represent a global fit of the mixed hyperbolic equation and showthe expected non-competitive inhibition of firefly luciferase activityby NFκBAI4. For comparison, see results obtained with resveratrolin [12]. (E) Decayof the firefly luciferase reaction during a 9 min time frameafter addition of coenzyme A-free reaction buffer in the presence orabsence of 1 μM NFκBAI4. The means of twoexperiments are given.
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Figure 3: Time-dependent firefly luciferase inhibition byNFκBAI4 in cell culture and analysis of the inhibitionmechanismLuciferase activity was assessed in lysates from various cell lines,derived from 70.4 or 55.1c mouse hepatoma cells, with stable expressionof firefly luciferase. Then 20 μM NFκBAI4 was addedto cell cultures and incubated for 1 h (A),24 h (B) or 48 h (C) prior tolysis and measurement. Luciferase signals were normalized to cellvitality, as determined by the Alamar Blue assay. Means±S.D.(n=3 and 4) are given;*P<0.05. For comparison see data forfirefly luciferase inhibition by resveratrol in Supplementary Figure S2at http://www.bioscirep.org/bsr/032/bsr0320531add.htm.(D) Dose-response analysis of firefly luciferaseactivity for the substrates D-luciferin and ATP in the presenceof different amounts of NFκBAI4. When varyingD-luciferin content of the reaction mixture, ATP was keptconstant at 500 μM; when varying ATP levels,D-luciferin was kept constant at 1000 μM. Solidlines represent a global fit of the mixed hyperbolic equation and showthe expected non-competitive inhibition of firefly luciferase activityby NFκBAI4. For comparison, see results obtained with resveratrolin [12]. (E) Decayof the firefly luciferase reaction during a 9 min time frameafter addition of coenzyme A-free reaction buffer in the presence orabsence of 1 μM NFκBAI4. The means of twoexperiments are given.

Mentions: In a series of experiments aimed at analysing a possible interplay ofβ-catenin signalling and other cellular signalling pathways, the murinehepatoma cell lines Hepa1c1c7, 70.4 and 55.1c were transiently transfected withthe β-catenin-driven firefly luciferase reporter STF and treated with20 μM NFκBAI4 for 24 h. As shown in Figure 1(B), NFκBAI4 treatment causedan unexpected strong decrease of luminescence in all three cell systems. Asimilar inhibitory effect of NFκBAI4 was observed when a number of stablySTF-transfected cell clones derived from the 70.4 or 55.1c cell lines weretreated with the compound (Figure 3B). Thiswas, however, not accompanied by a decrease in mRNA levels of the knownβ-catenin target genes Axin2 and Gpr49 (G-protein-coupled receptor 49)[15], thus casting doubt on a trueinhibition of the pathway (Figure 1C),especially as a considerable concomitant reduction of target gene expression andreporter activity can be achieved by transfection of siRNA (small interferingRNA) directed against β-catenin mRNA (results not shown). One possibleexplanation for this discrepancy of β-catenin-dependent fireflyluciferase reporter and target mRNA data was that NFκBAI4 causes aninhibition of the firefly luciferase reaction. To test this hypothesis,20 μM NFκBAI4 was added directly into lysates of untreatedHepa1c1c7, 70.4 and 55.1c cells which had been transiently transfected withexpression vectors for the firefly, Renilla orGaussia luciferase 24 h before. NFκBAI4strongly inhibited firefly luciferase signals in all cell lines, whereas theother luciferases, i.e. Gaussia and Renillaluciferase, were not affected (Figure 1D),demonstrating the specificity of NFκBAI4 for firefly luciferase. Purecommercially available firefly luciferase was also inhibited by NFκBAI4,demonstrating that the observed effects are not due to other components presentin cell lysates (Figure 1E). Inhibition offirefly luciferase activity was further monitored in livingluciferase-expressing cells incubated with a buffer containing thecell-permeable D-luciferin derivative D-luciferin-ethylester[20] following exposure of the cellsto NFκBAI4 (Figures 1F and 1G).


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

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

Time-dependent firefly luciferase inhibition byNFκBAI4 in cell culture and analysis of the inhibitionmechanismLuciferase activity was assessed in lysates from various cell lines,derived from 70.4 or 55.1c mouse hepatoma cells, with stable expressionof firefly luciferase. Then 20 μM NFκBAI4 was addedto cell cultures and incubated for 1 h (A),24 h (B) or 48 h (C) prior tolysis and measurement. Luciferase signals were normalized to cellvitality, as determined by the Alamar Blue assay. Means±S.D.(n=3 and 4) are given;*P<0.05. For comparison see data forfirefly luciferase inhibition by resveratrol in Supplementary Figure S2at http://www.bioscirep.org/bsr/032/bsr0320531add.htm.(D) Dose-response analysis of firefly luciferaseactivity for the substrates D-luciferin and ATP in the presenceof different amounts of NFκBAI4. When varyingD-luciferin content of the reaction mixture, ATP was keptconstant at 500 μM; when varying ATP levels,D-luciferin was kept constant at 1000 μM. Solidlines represent a global fit of the mixed hyperbolic equation and showthe expected non-competitive inhibition of firefly luciferase activityby NFκBAI4. For comparison, see results obtained with resveratrolin [12]. (E) Decayof the firefly luciferase reaction during a 9 min time frameafter addition of coenzyme A-free reaction buffer in the presence orabsence of 1 μM NFκBAI4. The means of twoexperiments are given.
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Related In: Results  -  Collection

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Figure 3: Time-dependent firefly luciferase inhibition byNFκBAI4 in cell culture and analysis of the inhibitionmechanismLuciferase activity was assessed in lysates from various cell lines,derived from 70.4 or 55.1c mouse hepatoma cells, with stable expressionof firefly luciferase. Then 20 μM NFκBAI4 was addedto cell cultures and incubated for 1 h (A),24 h (B) or 48 h (C) prior tolysis and measurement. Luciferase signals were normalized to cellvitality, as determined by the Alamar Blue assay. Means±S.D.(n=3 and 4) are given;*P<0.05. For comparison see data forfirefly luciferase inhibition by resveratrol in Supplementary Figure S2at http://www.bioscirep.org/bsr/032/bsr0320531add.htm.(D) Dose-response analysis of firefly luciferaseactivity for the substrates D-luciferin and ATP in the presenceof different amounts of NFκBAI4. When varyingD-luciferin content of the reaction mixture, ATP was keptconstant at 500 μM; when varying ATP levels,D-luciferin was kept constant at 1000 μM. Solidlines represent a global fit of the mixed hyperbolic equation and showthe expected non-competitive inhibition of firefly luciferase activityby NFκBAI4. For comparison, see results obtained with resveratrolin [12]. (E) Decayof the firefly luciferase reaction during a 9 min time frameafter addition of coenzyme A-free reaction buffer in the presence orabsence of 1 μM NFκBAI4. The means of twoexperiments are given.
Mentions: In a series of experiments aimed at analysing a possible interplay ofβ-catenin signalling and other cellular signalling pathways, the murinehepatoma cell lines Hepa1c1c7, 70.4 and 55.1c were transiently transfected withthe β-catenin-driven firefly luciferase reporter STF and treated with20 μM NFκBAI4 for 24 h. As shown in Figure 1(B), NFκBAI4 treatment causedan unexpected strong decrease of luminescence in all three cell systems. Asimilar inhibitory effect of NFκBAI4 was observed when a number of stablySTF-transfected cell clones derived from the 70.4 or 55.1c cell lines weretreated with the compound (Figure 3B). Thiswas, however, not accompanied by a decrease in mRNA levels of the knownβ-catenin target genes Axin2 and Gpr49 (G-protein-coupled receptor 49)[15], thus casting doubt on a trueinhibition of the pathway (Figure 1C),especially as a considerable concomitant reduction of target gene expression andreporter activity can be achieved by transfection of siRNA (small interferingRNA) directed against β-catenin mRNA (results not shown). One possibleexplanation for this discrepancy of β-catenin-dependent fireflyluciferase reporter and target mRNA data was that NFκBAI4 causes aninhibition of the firefly luciferase reaction. To test this hypothesis,20 μM NFκBAI4 was added directly into lysates of untreatedHepa1c1c7, 70.4 and 55.1c cells which had been transiently transfected withexpression vectors for the firefly, Renilla orGaussia luciferase 24 h before. NFκBAI4strongly inhibited firefly luciferase signals in all cell lines, whereas theother luciferases, i.e. Gaussia and Renillaluciferase, were not affected (Figure 1D),demonstrating the specificity of NFκBAI4 for firefly luciferase. Purecommercially available firefly luciferase was also inhibited by NFκBAI4,demonstrating that the observed effects are not due to other components presentin cell lysates (Figure 1E). Inhibition offirefly luciferase activity was further monitored in livingluciferase-expressing cells incubated with a buffer containing thecell-permeable D-luciferin derivative D-luciferin-ethylester[20] following exposure of the cellsto NFκBAI4 (Figures 1F and 1G).

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.

Show MeSH
Related in: MedlinePlus