Potential problems inherent in cell-based stable NF-κB–GFP reporter systems

El-Guendy, Nadia; Sinai, Æ Anthony P.

doi:10.1007/s11010-008-9730-8

Cited by 6 publications

(4 citation statements)

References 29 publications

(40 reference statements)

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“…Our global analysis of mRNA expression also revealed that care should be taken to use GFP-expressing vectors as a control as our results clearly reveal a dominant negative effect on Cxcl2 mRNA but also on ActB and Ube2l3 expression. This is in line with recent studies showing that GFP inhibits IL-2 expression in T cells and that NF-κB-driven GFP reporter constructs deteriorate expression of LPS- or TNF-induced endogenous IL6 and Cxcl1 genes ( 79 , 80 ). This might be due to defective polyubiquitinylation in GFP expressing cells which can perturb ubiquitin-based signaling cascades ( 81 ).…”

Section: Discussionsupporting

confidence: 91%

RNAi-Based Identification of Gene-Specific Nuclear Cofactor Networks Regulating Interleukin-1 Target Genes

et al. 2018

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The potent proinflammatory cytokine interleukin (IL)-1 triggers gene expression through the NF-κB signaling pathway. Here, we investigated the cofactor requirements of strongly regulated IL-1 target genes whose expression is impaired in p65 NF-κB-deficient murine embryonic fibroblasts. By two independent small-hairpin (sh)RNA screens, we examined 170 genes annotated to encode nuclear cofactors for their role in Cxcl2 mRNA expression and identified 22 factors that modulated basal or IL-1-inducible Cxcl2 levels. The functions of 16 of these factors were validated for Cxcl2 and further analyzed for their role in regulation of 10 additional IL-1 target genes by RT-qPCR. These data reveal that each inducible gene has its own (quantitative) requirement of cofactors to maintain basal levels and to respond to IL-1. Twelve factors (Epc1, H2afz, Kdm2b, Kdm6a, Mbd3, Mta2, Phf21a, Ruvbl1, Sin3b, Suv420h1, Taf1, and Ube3a) have not been previously implicated in inflammatory cytokine functions. Bioinformatics analysis indicates that they are components of complex nuclear protein networks that regulate chromatin functions and gene transcription. Collectively, these data suggest that downstream from the essential NF-κB signal each cytokine-inducible target gene has further subtle requirements for individual sets of nuclear cofactors that shape its transcriptional activation profile.

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Section: Discussionsupporting

confidence: 91%

RNAi-Based Identification of Gene-Specific Nuclear Cofactor Networks Regulating Interleukin-1 Target Genes

et al. 2018

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“…As shown previously by El-Guendy and colleagues, NFκB transcriptional activity can be measured by an NFκB reporter gene assay (El-Guendy and Sinai 2008 ). Stably transfected HEK 293 Nano-PEST cells showed an almost 25-fold increase in Nanoluciferase activity after stimulation with 25 ng/ml TNFα for 3 h. This increase could be significantly and dose-dependently decreased by 31.0, 34.7, and 42.1% after 8 h of pre-incubation with EA 575 ® concentrations of 40, 80, and 240 µg/ml, respectively (Fig.…”

Section: Resultsmentioning

confidence: 94%

Anti-inflammatory effects of ivy leaves dry extract: influence on transcriptional activity of NFκB

et al. 2018

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EA 575 is an ivy leaves dry extract (DER 5-7.5:1, 30% ethanol) used against diseases of the lower respiratory tract associated with productive cough. EA 575 improves symptoms associated with chronic inflammatory bronchial conditions. Compared to its bronchospasmolytic and secretolytic properties, the anti-inflammatory effects of EA 575 are mostly untried. Therefore, we addressed the question of whether the anti-inflammatory effect of EA 575 is due to an impact on the NFκB pathway. NFκB nuclear translocation was visualized by immunofluorescence in J774.2 as well as HEK293 cells. In the latter, a luciferase-based reporter was used to monitor NFκB transcriptional activity. Phosphorylation of RelA and its inhibitor IκB was measured by Western blot analysis. Additionally, changes in the stability of NFκB:IκB complex were shown by protein fragment complementation. Decreased transcriptional activity of NFκB under treatment with EA 575 was also shown for a human monocytic as well as a human lung epithelial cell line. EA 575 is able to inhibit NFκB transcriptional activity by partially inhibiting its translocation to the nucleus after stimulation with TNFα. Furthermore, phosphorylation of IκBα is reduced while phosphorylation of RelA is enhanced after pre-incubation with EA 575, leading to an enhanced stability of NFκB:IκBα complex. EA 575 has an regulatory impact on the NFκB pathway, possibly by switching specificity of IKK from IκBα to RelA, resulting in enhanced stability of NFκB:IκBα complex and reduced RelA translocation into the nucleus.

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“…Finally, to assess directly NF-κB transcriptional activity, we measured CXCL-1 (GRO-1) and CXCL-2 (GRO-2) mRNAs, broadly accepted as NF-κB target genes ( El-Guendy and Sinai, 2008 ; Patterson et al. , 2013 ).…”

Section: Resultsmentioning

confidence: 99%

Conditional knockout of polarity complex (atypical) PKCι reveals an anti-inflammatory function mediated by NF-κB

Forteza

Terán-Figueroa

Mashukova

et al. 2016

MBoC

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Potential problems inherent in cell-based stable NF-κB–GFP reporter systems

Cited by 6 publications

References 29 publications

RNAi-Based Identification of Gene-Specific Nuclear Cofactor Networks Regulating Interleukin-1 Target Genes

RNAi-Based Identification of Gene-Specific Nuclear Cofactor Networks Regulating Interleukin-1 Target Genes

Anti-inflammatory effects of ivy leaves dry extract: influence on transcriptional activity of NFκB

Conditional knockout of polarity complex (atypical) PKCι reveals an anti-inflammatory function mediated by NF-κB

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