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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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Dose–dependency of firefly luciferase inhibition byNFκBAI4 and resveratrolDose-effect curves of luciferase inhibition by NFκBAI4(A) or resveratrol (B) are shown inlysates from untreated 70.4STF K15 and 55.1cSTF K65 cells with stableexpression of firefly luciferase. Then 20 μMNFκBAI4 was added to lysates of untreated cells 5 minprior to measurement. Means±S.D. (n=4) aregiven. Inhibition of firefly luciferase activity in lysates derived fromother cell lines is depicted in Supplementary Figure S1 at http://www.bioscirep.org/bsr/032/bsr0320531add.htm.
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Figure 2: Dose–dependency of firefly luciferase inhibition byNFκBAI4 and resveratrolDose-effect curves of luciferase inhibition by NFκBAI4(A) or resveratrol (B) are shown inlysates from untreated 70.4STF K15 and 55.1cSTF K65 cells with stableexpression of firefly luciferase. Then 20 μMNFκBAI4 was added to lysates of untreated cells 5 minprior to measurement. Means±S.D. (n=4) aregiven. Inhibition of firefly luciferase activity in lysates derived fromother cell lines is depicted in Supplementary Figure S1 at http://www.bioscirep.org/bsr/032/bsr0320531add.htm.

Mentions: It has been reported that resveratrol, structurally related to NFκBAI4,inhibits firefly luciferase [6,12]. We thus compared the ability of bothcompounds to inhibit firefly luciferase activity derived from lysates ofuntreated firefly luciferase-expressing cells (Figure 2, and Supplementary Figure S1 at http://www.bioscirep.org/bsr/032/bsr0320531add.htm). Theinhibitory potency of both substances was very similar in all four cell linesanalysed, with IC50 values of ~1 μM (Table 1). Almost identicalconcentration-dependent inhibition (IC50≈1 μM)of firefly luciferase by NFκBAI4 was detected when a reaction bufferwithout coenzyme A was used (results not shown; for comparison, see also resultsin Figure 3E). Values obtained withresveratrol were comparable with a previously reported IC50 value of~2 μM [12].


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

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

Dose–dependency of firefly luciferase inhibition byNFκBAI4 and resveratrolDose-effect curves of luciferase inhibition by NFκBAI4(A) or resveratrol (B) are shown inlysates from untreated 70.4STF K15 and 55.1cSTF K65 cells with stableexpression of firefly luciferase. Then 20 μMNFκBAI4 was added to lysates of untreated cells 5 minprior to measurement. Means±S.D. (n=4) aregiven. Inhibition of firefly luciferase activity in lysates derived fromother cell lines is depicted in Supplementary Figure S1 at http://www.bioscirep.org/bsr/032/bsr0320531add.htm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475454&req=5

Figure 2: Dose–dependency of firefly luciferase inhibition byNFκBAI4 and resveratrolDose-effect curves of luciferase inhibition by NFκBAI4(A) or resveratrol (B) are shown inlysates from untreated 70.4STF K15 and 55.1cSTF K65 cells with stableexpression of firefly luciferase. Then 20 μMNFκBAI4 was added to lysates of untreated cells 5 minprior to measurement. Means±S.D. (n=4) aregiven. Inhibition of firefly luciferase activity in lysates derived fromother cell lines is depicted in Supplementary Figure S1 at http://www.bioscirep.org/bsr/032/bsr0320531add.htm.
Mentions: It has been reported that resveratrol, structurally related to NFκBAI4,inhibits firefly luciferase [6,12]. We thus compared the ability of bothcompounds to inhibit firefly luciferase activity derived from lysates ofuntreated firefly luciferase-expressing cells (Figure 2, and Supplementary Figure S1 at http://www.bioscirep.org/bsr/032/bsr0320531add.htm). Theinhibitory potency of both substances was very similar in all four cell linesanalysed, with IC50 values of ~1 μM (Table 1). Almost identicalconcentration-dependent inhibition (IC50≈1 μM)of firefly luciferase by NFκBAI4 was detected when a reaction bufferwithout coenzyme A was used (results not shown; for comparison, see also resultsin Figure 3E). Values obtained withresveratrol were comparable with a previously reported IC50 value of~2 μM [12].

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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