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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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Related in: MedlinePlus

Inhibition of firefly luciferase by the NF-κB inhibitorNFκBAI4(A) Chemical structures of NFκBAI4 and its analogueresveratrol. (B) Inhibition of firefly luciferase activity isobserved in mouse hepatoma cells transiently transfected with theβ-catenin-driven firefly luciferase reporter STF after 24 h ofincubation of the cells with 20 μM NFκBAI4. Luciferasesignals were normalized to cell vitality, as determined by the Alamar Blueassay. Means±S.D. (n=4) are given;*P<0.05. (C) Lack ofinhibition of the known β-catenin target genes Axin2 and Gpr49 by24 h treatment of cells with 20 μM NFκBAI4.Means±S.D. (n=6) are given. (D)Inhibition of firefly luciferase signals after addition of20 μM NFκBAI4 [1% (v/v) of 2 mM NFκBAI4solution in DMSO] to lysates of untreated, transiently luciferase-expressingHepa1c1c7, 70.4 and 55.1c cells 5 min prior to measurement. Noinhibition of Gaussia or Renillaluciferases is observed. Means±S.D. (n=3 and 4) aregiven; *P<0.05. (E) Inhibitionof commercially available firefly luciferase by 10 μMNFκBAI4 at different concentrations of the enzyme. The means of twoexperiments are shown. (F) Inhibition of firefly luciferaseactivity in living stably luciferase-expressing cells (cell lines 70.4 K15and 55.1c K65) by NFκBAI4. Cells were pre-incubated with20 μM NFκBAI4 for 30 min prior to analysis.Means±S.D. (n=4) are given;*P<0.05. (G) Luciferaseinhibition in living cells was also monitored using a CCD camera system. Arepresentative image from 55.1c K65 cells is shown. (H)Inhibition of luminescence signals from the CellTiter-Glo cell viabilityassay kit, which is based on luciferase-dependent detection of cellular ATP,by addition of 200 μM NFκBAI4. Please note that200 μM NFκBAI4 is needed for ~50% inhibition ofthe Ultra-Glo luciferase used in this assay. Means±S.D.(n=3–5, each experiment performed in eightdeterminations) are given; *P<0.05.
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Figure 1: Inhibition of firefly luciferase by the NF-κB inhibitorNFκBAI4(A) Chemical structures of NFκBAI4 and its analogueresveratrol. (B) Inhibition of firefly luciferase activity isobserved in mouse hepatoma cells transiently transfected with theβ-catenin-driven firefly luciferase reporter STF after 24 h ofincubation of the cells with 20 μM NFκBAI4. Luciferasesignals were normalized to cell vitality, as determined by the Alamar Blueassay. Means±S.D. (n=4) are given;*P<0.05. (C) Lack ofinhibition of the known β-catenin target genes Axin2 and Gpr49 by24 h treatment of cells with 20 μM NFκBAI4.Means±S.D. (n=6) are given. (D)Inhibition of firefly luciferase signals after addition of20 μM NFκBAI4 [1% (v/v) of 2 mM NFκBAI4solution in DMSO] to lysates of untreated, transiently luciferase-expressingHepa1c1c7, 70.4 and 55.1c cells 5 min prior to measurement. Noinhibition of Gaussia or Renillaluciferases is observed. Means±S.D. (n=3 and 4) aregiven; *P<0.05. (E) Inhibitionof commercially available firefly luciferase by 10 μMNFκBAI4 at different concentrations of the enzyme. The means of twoexperiments are shown. (F) Inhibition of firefly luciferaseactivity in living stably luciferase-expressing cells (cell lines 70.4 K15and 55.1c K65) by NFκBAI4. Cells were pre-incubated with20 μM NFκBAI4 for 30 min prior to analysis.Means±S.D. (n=4) are given;*P<0.05. (G) Luciferaseinhibition in living cells was also monitored using a CCD camera system. Arepresentative image from 55.1c K65 cells is shown. (H)Inhibition of luminescence signals from the CellTiter-Glo cell viabilityassay kit, which is based on luciferase-dependent detection of cellular ATP,by addition of 200 μM NFκBAI4. Please note that200 μM NFκBAI4 is needed for ~50% inhibition ofthe Ultra-Glo luciferase used in this assay. Means±S.D.(n=3–5, each experiment performed in eightdeterminations) are given; *P<0.05.

Mentions: In the present study, we demonstrate that (E)-2-fluoro-4′-methoxystilbene{also known as NFκBAI4 [NF-κB (nuclear factor κB) activationinhibitor 4]; Figure 1A}, a resveratrolanalogue without antioxidative properties used as a specific inhibitor ofNF-κB activation [13], inhibitsfirefly luciferase, but not other luciferases, in vitro with apotency comparable with resveratrol. In contrast with resveratrol, inhibition offirefly luciferase by NFκBAI4 is sustained for >24 h in livingcells, much longer than inhibition by resveratrol.


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

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

Inhibition of firefly luciferase by the NF-κB inhibitorNFκBAI4(A) Chemical structures of NFκBAI4 and its analogueresveratrol. (B) Inhibition of firefly luciferase activity isobserved in mouse hepatoma cells transiently transfected with theβ-catenin-driven firefly luciferase reporter STF after 24 h ofincubation of the cells with 20 μM NFκBAI4. Luciferasesignals were normalized to cell vitality, as determined by the Alamar Blueassay. Means±S.D. (n=4) are given;*P<0.05. (C) Lack ofinhibition of the known β-catenin target genes Axin2 and Gpr49 by24 h treatment of cells with 20 μM NFκBAI4.Means±S.D. (n=6) are given. (D)Inhibition of firefly luciferase signals after addition of20 μM NFκBAI4 [1% (v/v) of 2 mM NFκBAI4solution in DMSO] to lysates of untreated, transiently luciferase-expressingHepa1c1c7, 70.4 and 55.1c cells 5 min prior to measurement. Noinhibition of Gaussia or Renillaluciferases is observed. Means±S.D. (n=3 and 4) aregiven; *P<0.05. (E) Inhibitionof commercially available firefly luciferase by 10 μMNFκBAI4 at different concentrations of the enzyme. The means of twoexperiments are shown. (F) Inhibition of firefly luciferaseactivity in living stably luciferase-expressing cells (cell lines 70.4 K15and 55.1c K65) by NFκBAI4. Cells were pre-incubated with20 μM NFκBAI4 for 30 min prior to analysis.Means±S.D. (n=4) are given;*P<0.05. (G) Luciferaseinhibition in living cells was also monitored using a CCD camera system. Arepresentative image from 55.1c K65 cells is shown. (H)Inhibition of luminescence signals from the CellTiter-Glo cell viabilityassay kit, which is based on luciferase-dependent detection of cellular ATP,by addition of 200 μM NFκBAI4. Please note that200 μM NFκBAI4 is needed for ~50% inhibition ofthe Ultra-Glo luciferase used in this assay. Means±S.D.(n=3–5, each experiment performed in eightdeterminations) are given; *P<0.05.
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Related In: Results  -  Collection

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Figure 1: Inhibition of firefly luciferase by the NF-κB inhibitorNFκBAI4(A) Chemical structures of NFκBAI4 and its analogueresveratrol. (B) Inhibition of firefly luciferase activity isobserved in mouse hepatoma cells transiently transfected with theβ-catenin-driven firefly luciferase reporter STF after 24 h ofincubation of the cells with 20 μM NFκBAI4. Luciferasesignals were normalized to cell vitality, as determined by the Alamar Blueassay. Means±S.D. (n=4) are given;*P<0.05. (C) Lack ofinhibition of the known β-catenin target genes Axin2 and Gpr49 by24 h treatment of cells with 20 μM NFκBAI4.Means±S.D. (n=6) are given. (D)Inhibition of firefly luciferase signals after addition of20 μM NFκBAI4 [1% (v/v) of 2 mM NFκBAI4solution in DMSO] to lysates of untreated, transiently luciferase-expressingHepa1c1c7, 70.4 and 55.1c cells 5 min prior to measurement. Noinhibition of Gaussia or Renillaluciferases is observed. Means±S.D. (n=3 and 4) aregiven; *P<0.05. (E) Inhibitionof commercially available firefly luciferase by 10 μMNFκBAI4 at different concentrations of the enzyme. The means of twoexperiments are shown. (F) Inhibition of firefly luciferaseactivity in living stably luciferase-expressing cells (cell lines 70.4 K15and 55.1c K65) by NFκBAI4. Cells were pre-incubated with20 μM NFκBAI4 for 30 min prior to analysis.Means±S.D. (n=4) are given;*P<0.05. (G) Luciferaseinhibition in living cells was also monitored using a CCD camera system. Arepresentative image from 55.1c K65 cells is shown. (H)Inhibition of luminescence signals from the CellTiter-Glo cell viabilityassay kit, which is based on luciferase-dependent detection of cellular ATP,by addition of 200 μM NFκBAI4. Please note that200 μM NFκBAI4 is needed for ~50% inhibition ofthe Ultra-Glo luciferase used in this assay. Means±S.D.(n=3–5, each experiment performed in eightdeterminations) are given; *P<0.05.
Mentions: In the present study, we demonstrate that (E)-2-fluoro-4′-methoxystilbene{also known as NFκBAI4 [NF-κB (nuclear factor κB) activationinhibitor 4]; Figure 1A}, a resveratrolanalogue without antioxidative properties used as a specific inhibitor ofNF-κB activation [13], inhibitsfirefly luciferase, but not other luciferases, in vitro with apotency comparable with resveratrol. In contrast with resveratrol, inhibition offirefly luciferase by NFκBAI4 is sustained for >24 h in livingcells, much longer than inhibition by resveratrol.

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