A high-sensitivity, bi-directional reporter to monitor NF-κB activity in cell culture and zebrafish in real-time

Abstract

NF-κB transcription factors play major roles in numerous biological processes including development and immunity. Here, we engineered a novel bi-directional NF-κB-responsive reporter, pSGNluc, in which a high-affinity NF-κB promoter fragment simultaneously drives expression of luciferase and GFP. Treatment with TNFα induced a strong, dose-dependent luciferase signal in cell culture. The degree of induction over background was comparable to that of other NF-κB-driven luciferase reporters, but the absolute level of expression was at least 20-fold higher. This extends the sensitivity range of otherwise difficult assays mediated exclusively by endogenously expressed receptors, as we show for Nod1 signalling in HEK293 cells. To measure NF-κB activity in the living organism, we established a transgenic zebrafish line carrying the pSGNluc construct. Live in toto imaging of transgenic embryos revealed the activation patterns of NF-κB signalling during embryonic development and as responses to inflammatory stimuli. Altogether, by integrating qualitative and quantitative NF-κB reporter activity, pSGNluc is a valuable tool for studying NF-κB signaling at high spatiotemporal resolution in cultured cells and living animals which goes beyond the possibilities provided by currently available reporters.

Full text

TOOLS AND TECHNIQUES
A high-sensitivity bi-directional reporter to monitor NF-κB activity
in cell culture and zebrafish in real time
Paola Kuri
1
, Kornelia Ellwanger
2
, Thomas A. Kufer
2
, Maria Leptin
1,3,4,
*and Baubak Bajoghli
1,
*
ABSTRACT
Nuclear factor (NF)-κB transcription factors play major roles in
numerous biological processes including development and immunity.
Here, we engineered a novel bi-directional NF-κB-responsive
reporter, pSGNluc, in which a high-affinity NF-κB promoter
fragment simultaneously drives expression of luciferase and GFP.
Treatment with TNFα(also known as TNF) induced a strong, dose-
dependent luciferase signal in cell culture. The degree of induction
over background was comparable to that of other NF-κB-driven
luciferase reporters, but the absolute level of expression was at least
20-fold higher. This extends the sensitivity range of otherwise difficult
assays mediated exclusively by endogenously expressed receptors,
as we show for Nod1 signaling in HEK293 cells. To measure NF-κB
activity in the living organism, we established a transgenic zebrafish
line carrying the pSGNluc construct. Live in toto imaging of transgenic
embryos revealed the activation patterns of NF-κB signaling during
embryonic development and as responses to inflammatory stimuli.
Taken together, by integrating qualitative and quantitative NF-κB
reporter activity, pSGNluc is a valuable tool for studying NF-κB
signaling at high spatiotemporal resolution in cultured cells and living
animals that goes beyond the possibilities provided by currently
available reporters.
KEY WORDS: Nod1, Tri-DAP, pSGNluc, Proctodeum, Zebrafish
INTRODUCTION
Nuclear factor (NF)-κB transcription factors are essential for a
number of biological processes such as inflammatory and immune
responses, cell growth, apoptosis and development. Their
inappropriate activation has been linked to autoimmunity, chronic
inflammation and various types of cancer (Ben-Neriah and Karin,
2011; Hayden and Ghosh, 2008; Napetschnig and Wu, 2013).
NF-κB transcription factors are expressed in many cell types.
Five NF-κB polypeptides, p65 (RelA), c-Rel, RelB, p50 (encoded
by NFKB1) and p52 (encoded by NFKB2), can combine to form 15
different transcription factors through homo- and hetero-
dimerization (Hoffmann and Baltimore, 2006). The transcriptional
activation of target genes depends on the nuclear translocation of
NF-κB dimers. In the absence of stimulatory signals, inhibitory κB
protein family members (e.g. IκBαand IκBβ, also known as
NFKBIA and NFKBIB, respectively) keep NF-κB transcription
factors sequestered in the cytoplasm, thereby preventing their
binding to DNA. Activation of different signaling pathways by
proinflammatory signals such as pathogen-associated molecular
patterns, danger-associated molecular patterns or proinflammatory
cytokines (e.g. interleukin-1), results in IκB kinase (IKK)
activation. This kinase complex phosphorylates the IκBs,
inducing their proteasomal degradation and resulting in release of
NF-κB transcription factors and their translocation into the nucleus
(Vallabhapurapu and Karin, 2009). All NF-κB transcription factors
share the ability to bind to the κB site consensus sequence 5′-
GGGRNWYYCC-3′(where R is a purine, Y is a pyrimidine, W is
an adenine or thymine and N is an unspecified base) (Hoffmann and
Baltimore, 2006). However, the binding affinity of NF-κB
transcription factors to different κB site sequences varies,
indicating a relationship between binding affinity, specificity and
function (Siggers et al., 2012).
Various assays have been established to determine the activity of
the canonical NF-κB pathway. For example, IκBαdegradation
assayed by western blotting, and analysis of NF-κB binding activity
in the absence and presence of IκBαby electrophoretic mobility
shift assay (Vancurova et al., 2001). However, these time-
consuming methods are restricted to the analysis of small sample
numbers. An IκBα-firefly luciferase fusion reporter (IκBα-FLuc)
allows IKK activation to be monitored in real time (Gross and
Piwnica-Worms, 2005). Non-canonical NF-κB activation cannot be
measured using these methods since it occurs independently of
IκBαdegradation (Hayden and Ghosh, 2004). To assess NF-κB
transcription factor dynamics, cell lines that stably express the NF-
κB subunit p65 fused to GFP (Bartfeld et al., 2010) are used to
quantify the nuclear translocation of p65 in response to stimulation.
These assays, however, do not provide direct levels of downstream
transcriptional activation by NF-κB. The more commonly used
method to assay NF-κB activation is based on reporter genes (e.g.
luciferase or β-galactosidase) driven by promoters containing
multiple repeats of an NF-κB consensus sequence (Badr et al.,
2009; Bowie and O’Neill, 2000; Matsuda et al., 2007; Munoz et al.,
1994; Schindler and Baichwal, 1994). The sensitivity and
specificity for NF-κB activation varies between these reporters. In
this work, we have used a palindromic κB site sequence with high
binding affinity for multiple NF-κB proteins and developed a new
bi-directional NF-κB-responsive promoter to simultaneously drive
the GFP and luciferase reporter genes. We named this reporter
pSGNluc. We found that pSGNluc is highly sensitive and inducible
in response to various stimuli in cell culture. In addition, we
established a transgenic zebrafish line carrying the pSGNluc
reporter. We show that GFP and luciferase reporters can be used
for different purposes, allowing us to visualize and quantify the NF-
κB activity during embryonic development and in response to
inflammation in live embryos. In toto imaging of zebrafish embryos
carrying pSGNluc revealed a dynamic NF-κB activity during early
development. Our data also suggest that Iκbαa (encoded by
Received 12 September 2016; Accepted 8 December 2016
1
Directors’Research Unit, European Molecular Biology Laboratory (EMBL),
Meyerhofstrasse 1, 69117 Heidelberg, Germany.
2
Department of Immunology,
Institute of Nutritional Medicine, Universityof Hohenheim, 70593 Stuttgart, Germany.
3
Institute of Genetics, University of Cologne, Zu
̈lpicherstrasse 47a, 50674 Cologne,
Germany.
4
EMBO, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
*Authors for correspondence (bajoghli@embl.de; maria.leptin@e mbo.org)
B.B., 0000-0002-7368-7523
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© 2017. Published by The Company of Biologists Ltd
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Journal of Cell Science (2017) 130, 648-657 doi:10.1242/jcs.196485
Journal of Cell Science

nfkbiaa), a member of the family of NF-κB inhibitory proteins,
regulates the NF-κB activity in the proctodeum and skin.
RESULTS
Design of the pSGNluc reporter
Most available NF-κB-responsive reporters use multimerized κB
site sequences upstream of a minimal promoter to drive a firefly
luciferase gene. The sequence and number of κB sites vary between
different NF-κB reporters. According to a large dataset of potential
κB site sequences for multiple NF-κB dimers (Siggers et al., 2012),
the predicted binding affinity of NF-κB dimers to the κB site
sequences used to generate these NF-κB reporters is moderate
(Fig. 1A). We hypothesized that a binding site with high affinity for
a range of different NF-κB dimers might allow us to make a more
sensitive reporter. The study by Siggers et al. showed that the
sequence 5′-GGGAATTCCC-3′, originally identified as a κB site
upstream of the type VII collagen gene (Kon et al., 1999), is able to
interact strongly with different combinations of hetero- and homo-
dimers of NF-κB subunits. In designing a new reporter plasmid, we
took advantage of the palindromic nature of this sequence to create a
bi-directional reporter. In this plasmid, which we named pSGNluc,
two minimal promoters flank a DNA fragment containing eight
tandem copies of the 5′-GGGAATTCCC-3′sequence (Fig. 1B).
GFP was cloned downstream of one promoter to visualize
expression in living cells. To enable quantitative analysis of
expression levels, luciferase was cloned downstream of the minimal
promoter on the opposing side (Fig. 1C). The pSGNluc reporter
should therefore simultaneously express both marker genes from the
bi-directional promoter, allowing both the spatial and temporal
monitoring and quantification of NF-κB activity in living cells.
The pSGNluc reporter is strongly inducible and highly
sensitive in cell culture
To examine the efficiency of the new NF-κB-responsive reporter in
cell culture, we transfected the pSGNluc vector in HEK293T cells.
Transfected, unstimulated cells showed weak or no GFP expression
(Fig. 2A), indicating a low background activity of the pSGNluc
reporter. Exposure to 10 ng/ml of TNFα(also known as TNF) led to
a strong upregulation of GFP expression in transfected cells, as
shown by live-cell microscopy and flow cytometry data (Fig. 2A,B).
We also measured the level of luciferase activity of pSGNluc,
and compared it to two other NF-κB-driven luciferase reporters,
pGL4.32 (Promega) and IgκB-Luci (Munoz et al., 1994) as well as
an IL-8 reporter (Bowie et al., 2000). We treated cells transfected
with each of these constructs with 10 ng/ml of TNFαand measured
the luciferase activity in cell lysates 24 h after treatment. We
calculated the inducibility of each of the reporters (measured as the
ratio of luciferase with and without treatment) and observed that
pSGNluc was induced to the same extent as pGL4.32 when
compared to non-treated controls. The IgκB-Luci and IL8-Luc
reporters displayed the highest and lowest inducibility levels,
respectively (Fig. 2C). All transfected cells showed low levels of
background signal without stimulation, although the pSGNluc
reporter was expressed at up to 6-fold higher levels than the IL-8
reporter (Table S1). To compare the sensitivity of the NF-κB-
responsive reporters, the cells were stimulated with increasing
TNFαconcentrations. The luciferase level of all four reporters
increased in a TNFα-dose-dependent manner after a 24 h treatment.
The absolute level of luciferase expressed with the pSGNluc
reporter in response to TNFαat 1 ng/ml was 21-, 34- and 73-fold
higher than for the IgκB-Luci, pGL4.32 and IL-8 reporters
(Fig. 2D,E).
We then determined the kinetics of NF-κB activation in response
to 10 ng/ml TNFα. The pSGNluc reporter reached luciferase
activity levels that were significantly higher than other reporters
tested from 6 h after treatment (Fig. 2F), illustrating an increased
sensitivity even at early time points after stimulation.
We also tested the response of the reporter to other inflammatory
signals. Nod1 is an intracellular pattern recognition receptor that
induces NF-κB activation upon detection of bacterial
peptidoglycans (Chamaillard et al., 2003; Girardin et al., 2003).
HEK293T cells have low levels of endogenous Nod1 (Viala et al.,
2004). In many studies, HEK293T cells are therefore transfected
with Nod1 expression plasmids (Masumoto et al., 2006; Zurek
et al., 2011) to increase the NF-κB response towards Nod1
stimulation. However, the amount of Nod1 receptor in the cell is
crucial and its overexpression can result in autoactivation (Zurek
et al., 2011). We tested whether the high sensitivity of pSGNluc
would allow the detection of NF-κB activation mediated by the
endogenously expressed Nod1. HEK293T cells were transfected
with pSGNluc and then treated with L-Ala-γ-D-Glu-meso-
diaminopimelic acid (Tri-DAP) (Chamaillard et al., 2003;
Girardin et al., 2003). The levels of luciferase activity reached by
pSGNluc after Tri-DAP stimulation were 13- to 55-fold higher than
those of the pGL4.32, IgκB-Luci and IL-8 reporters (Fig. 3A;
Table S2), representing a 24±8-fold (mean±s.d.) increase in
luciferase activity in cells carrying the pSGNluc reporter
(Fig. 3B). The fact that these inductions were small compared to
those achieved by TNFαmight suggest that Tri-DAP is a weaker
activator of NF-κB signaling. However, when we looked at the GFP
expression pattern of transfected cells we observed that, instead of a
homogeneous low expression level that could be expected from
general weak response, only a fraction of transfected cells responded
to Tri-DAP stimulation (Fig. 3C,D). Time-lapse imaging revealed
that these cells expressed GFP at similar levels to cells treated with
TNFα(Fig. 3E; Movie 1), albeit with a delayed onset. This indicates
that the average level of luciferase activity in all cells after treatment
was low not because the Nod1 ligand Tri-DAP elicits a weak
response, but because only a limited number of cells responded to
this treatment.
Overall, cell culture assays showed that pSGNluc is inducible
after treatment with inflammatory stimulants to a degree that was
comparable with other reporters (measured as fold induction over
background expression), but is much more sensitive than other NF-
κB-driven luciferase reporters. In particular, we found pSGNluc
suitable to monitor NF-κB activation mediated by endogenous
Nod1 in HEK293T cells. Furthermore, the bi-directional promoter
allows the concomitant monitoring of NF-κB activation by
expression of the two reporters, GFP and luciferase, and enables
single-cell analysis of NF-κB activation and easier time-resolved
analysis.
Establishment of pSGNluc transgenic zebrafish to monitor
NF-κB activity in vivo
Members of the NF-κB transcription factor family are expressed
almost ubiquitously during embryonic development. In transgenic
mice carrying an NF-κB-responsive reporter, the spatial activity of
NF-κB signaling has been observed either in fixed tissues (Schmidt-
Ullrich et al., 1996) or in live animals with low spatiotemporal
resolution (Carlsen et al., 2002). Given that zebrafish NF-κB
proteins can bind to the mammalian consensus κB site (Correa et al.,
2004), we created transgenic zebrafish carrying the pSGNluc
reporter to observe NF-κB activity in the living organism. This
transgenic fish line is hereafter called Tg(8×Hs.NFκB:GFP,
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Luciferase) in accordance with the Zebrafish nomenclature
guidelines. We used light-sheet microscopy to monitor the
activity of the pSGNluc reporter during early zebrafish embryonic
development at high spatiotemporal resolution. Embryos carrying
the transgene expressed GFP in a dynamic pattern. Live in toto
imaging of the transgenic embryos from mid-gastrula to the
beginning of the segmentation stage [approximately from 6 to
12 hours post-fertilization (hpf )] revealed that there already was
ubiquitous and weak GFP expression at the shield stage, with the
entire animal pole labeled (Fig. 4A). Cells retained this level of
expression throughout the gastrula stage and into the segmentation
stage (Fig. 4B–E). The first increase in the GFP signal was observed
in the nasal vesicle (Fig. 4F) and in the proctodeum beginning
shortly afterwards (Fig. 4G). Using confocal microscopy, we
observed strong GFP expression in microvillous sensory neurons
that are connected to the forebrain (Fig. 4H), which diminished by
2 days post-fertilization (dpf ) (Fig. 4I). At 2 dpf, the signal was
strongly present in the lateral line (Fig. 4I, yellow arrows), the
proctodeum and epithelial cells around the edges of the dorsal and
ventral fins. This expression pattern remained at 3 dpf (Fig. 4J). By
5 dpf, cells in the intestinal lining began to express GFP, and
expression in the lateral line and fins remained (Fig. 4K, white
Fig. 1. The pSGNluc reporter plasmid.
(A) Z-score for κB site sequences present
in the IL-8-Luc, IgκB-Luci, pGL4.32 and
pSGNluc plasmids for their binding to
multiple NF-κB dimers; data are from the
protein-binding microarray dataset in
Siggers et al. (2012). (B) Sequence of
DNA fragment containing eight tandem
copies of the κB site (black boxes).
(C) Schematic representation of the
pSGNluc vector showing the eight copies
of the κB site (black), two minimal CMV
promoters (gray), GFP (green) and
luciferase (brown). The construct contains
two I-SceI meganuclease restriction sites
for the generation of transgenic animals.
Fig. 2. High sensitivity and inducibility of the pSGNluc reporter in response to TNFαstimulation. (A,B) HEK293T cells were transfected with the pSGNluc
and mCherry-expressing plasmids and treated with TNFα(10 ng/ml) where indicated. 24 h later, fluorescence signals were examined by fluorescence
microscopy (A) and flow cytometry (B). Expression of mCherry indicates the transfection efficiency. (C–F) HEK293T cells were transfected with the indicated
reporter plasmids and a β-galactosidase plasmid for normalization. Directly after transfection cellswere either left untreated or stimulated with TNFα(10 ng/ml). The
nRLU levels were measured 24 h later (see Materials and Methods). (C) The induction was calculated by dividing nRLU values from cells 24 h post stimulation
with TNFα(10 ng/ml) by nRLU values of unstimulated cells. Each dot represents the mean induction of independent experiments conducted in triplicates. (D)
Sensitivity matrix of NF-κB-responsive reporters based on nRLU upregulation after 24 h stimulation with TNFα(1 ng/ml). (E) TNFαtitration and (F) kinetics of
10 ng/ml TNFα-induced reporter activation. Values represent mean±s.e.m. (n=3). Scale bars: 80 µm. n.s., not significant.
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arrowheads). The onset of expression in the gut coincides with
the stage in which the larva begins to feed. After the onset of
hematopoiesis, embryos showed a dim GFP signal. A similar level
of GFP was also observed in keratinocytes. Zebrafish embryos
carrying a previously published NF-κB-responsive reporter also
expressed GFP in the skin (Candel et al., 2014; Hatzold et al., 2016).
However, the signal in the skin was stronger in embryos carrying the
latter reporter than in Tg(8×Hs.NFκB:GFP,Luciferase) embryos
(Fig. S1A). In mice, NF-κB proteins are mainly present in basal
keratinocytes (Takao et al., 2003), which are mitotically active and
provide new cells that gradually undergo differentiation toward the
skin surface. To better characterize the GFP signal in the skin, we
took advantage of the Tg(krt19:dTomato) fish, in which basal
keratinocytes are fluorescently labeled (Fischer et al., 2014). In vivo
imaging of double-transgenic Tg(8×Hs.NFκB:GFP,Luciferase;
krt19:dTomato) embryos revealed that GFP is expressed at a low
level in basal keratinocytes, albeit in a mosaic fashion (Fig. S2). As
NF-κB signaling has more than one role in keratinocytes, it remains
to be determined whether this expression pattern is associated with
keratinocyte homeostasis or inflammation.
To test the specificity of pSGNluc-driven GFP expression as a
proxy for NF-κB activation in zebrafish embryos, we interfered with
IκBαfunction. IκBα,anNF-κB inhibitory protein, binds to NF-κB
dimers and prevents their translocation into the nucleus (Arenzana-
Seisdedos et al., 1997). Knockdown of the zebrafish ortholog iκbαa
should therefore enable NF-κB proteins to move to the nucleus and
activate transcription of target genes (He et al., 2015). In zebrafish,
iκbαais expressed in the proctodeum and nasal vesicle (Fig. S3).
We injected a previously published morpholino (MO) (He et al.,
2015) to interfere with Iκbαa function in Tg(8×Hs.NFκB:GFP,
Luciferase) embryos. In iκbαamorphants, GFP expression was
enhanced in the skin and proctodeum (Fig. 5A–C). Although our
RNA in situ hybridization analysis did not detect iκbαaexpression
in the skin, previous microarray analysis has shown that both the
iκbαaand iκbαb(also known as nfkbiab) genes are expressed in the
zebrafish skin (Lü et al., 2012). At high doses, injection of the iκbαa
morpholino led to severe defects in notochord formation (Fig. 5D).
This phenotype is consistent with those previously observed in
zebrafish for NF-κB loss- or gain-of-function analysis (Correa et al.,
2005, 2004). The tissue-specific increase in GFP levels resulting
from knockdown of the NF-κB inhibitor iκbαasuggests that
reporter activity in the Tg(8×Hs.NFκB:GFP,Luciferase) line is
linked to NF-κB activation, and that the dynamic activity of NF-κB
signaling in various tissues during embryonic development can be
monitored through live imaging of this transgenic line.
In vivo monitoring of NF-κB activation in response to
inflammation
As regulators of the immune and inflammatory processes, NF-κB
transcription factors are activated in response to a variety of signals
including pathogens, stresses and injuries. We therefore monitored
the dynamics of NF-κB activation in response to injuries in
Fig. 3. Activation of the pSGNluc reporter in response to Nod1-mediated peptidoglycan recognition. (A,B) HEK293T cells were transfected with the
indicated reporter plasmids and a β-galactosidase plasmid for normalization. Subsequently cells were either left untreated or stimulated with 10 µg/ml Tri-DAP.
nRLU values were determined 24 h later (see Materials and Methods). (A) Tri-DAP (10 µg/ml)-induced reporter activation. Values represent mean±s.e.m. (n=3).
(B) The induction was calculated by dividing nRLU values of stimulated cells by control cells values. Each dot represents the mean induction of independent
experiments conducted in triplicates and the overall mean±s.d. is shown. (C) Representative images of HEK293T cells transfected with the pSGNluc and
mCherry-expressing plasmids. Expression of mCherry indicates the transfection efficiency. (D) GFP expression in cells after Tri-DAP stimulation (mean±s.e.m.;
n=3). The data from Fig. 2B and this figure come from the same experiment and the control group in the top panel of Fig. 2B is therefore also the control for this
panel. (E) Time course of GFP expression in pSGNluc-t ransfected cells without stimulation (top panel ), and cells treated with Tri-DAP at 10 µg/ml (middle panel)
or TNFαat 5 ng/ml (bottom panel). Images are taken from Movie 1. Red arrows indicate the onset of GFP expression. Scale bars: 80 µm (C); 100 µm (E).
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Tg(8×Hs.NFκB:GFP,Luciferase) embryos. In a tailfin-wounding
assay, GFP expression was upregulated in the area around the
wound edge of injured embryos (Fig. 6A; Movie 2). Most epithelial
cells at the wound edge exhibited strong NF-κB activation when
examined 24 h later. To test the inducibility of the reporter, we first
compared the kinetics of NF-κB activation in this assay with those
for a previously developed reporter in zebrafish (Banerjee and
Leptin, 2014). The Tg(NFκB-EGFP) line, previously used to
monitor NF-κB activation upon UV radiation in zebrafish embryos,
contains three copies of κB site sequences derived from the IgκB-
Luci vector (Banerjee and Leptin, 2014). No GFP is detectable
in Tg(NFκB-EGFP) embryos during embryonic development. In
response to sterile injury, Tg(NFκB-EGFP) embryos showed weak
GFP expression 40 h after wounding. In contrast, Tg(8×Hs.NFκB:
GFP,Luciferase) embryos showed a strong increase in GFP
expression 40 h after injury (Fig. S1B). Thus, Tg(8×Hs.NFκB:
GFP,Luciferase) embryos show a more sensitive response to local
injuries than the Tg(NFκB-EGFP).
We also tested the efficiency of the pSGNluc reporter in response
to infection. Non-pathogenic Gram-negative Escherichia coli
expressing red fluorescent protein were injected into the
notochord or the inner ear. No GFP expression driven by
pSGNluc was detectable at 3 dpf either in the notochord
(Fig. 6B, upper panel) or the inner ear (Fig. 6C, left panel) of
untreated animals. Injection of PBS alone resulted in GFP
expression in the notochord (Fig. 6B, middle panel) indicating
that a sterile injury alone activates NF-κB signaling. However, even
higher GFP levels were observed in notochords infected with
E. coli when examined 18 h post infection (Fig. 6B, bottom panel).
This is in agreement with previous studies showing that
microinjection of bacteria in these tissues results in an
inflammatory response that includes the recruitment of leukocytes
(Levraud et al., 2008; Nguyen-Chi et al., 2014). We also observed
an accumulation of GFP-expressing immune cells (Fig. 6C, right
Fig. 4. Dynamic NF-κB activity during early zebrafish embryonic
development. (A–E) Light-sheet images of Tg(8×Hs.NFκB:GFP,Luciferase)
embryos show ubiquitous, weak NF-κB activity from 6 hpf until 12 hpf. (F) The
first strong tissue-specific signal was observed in the nasal vesicle (nv) at
around 16 hpf. (G–K) Confocal images of Tg(8×Hs.NFκB:GFP,Luciferase)
embryos from 1 dpf until 5 dpf. GFP was detected in the proctodeum (p) at
1 dpf (G). The GFP signal in the nasal vesicle (G) originates from microvillous
sensory neurons (H). E–G and I–K show embryos in lateral view. Anterior is to
the left. H shows the head of embryos from the frontal side. Yellow arrows
indicate GFP in the lateral line. White arrowheads indicate GFP in the intestine.
Scale bars: 100 µm (A–F); 300 µm (G); 50 µm (H); 500 µm (I–K).
Fig. 5. Specificity of Tg(8xHs.NFκB:GFP,Luciferase) for NF-κB activity
in zebrafish embryos. (A,B) 0.3 mM iκbαamorpholino was injected into
transgenic embryos. Morphants at 3 dpf had increased expression of GFP
in the skin (A) and proctodeum (B, arrows). GFP fluorescence is shown as a
false-color heat map. The intensity of the laser was equal for both images to
allow direct comparison of the GFP signal in morphants and controls.
(C) Quantitative comparison of the GFP signal in the proctodeum of embryos
injected with 0.3 mM standard control or iκbαamorpholino. Each dot
represents an individual embryo at 2 dpf (mean±s.d.). (D) Injection of 1 mM
iκbαamorpholino results in higher GFP expression and embryonic
malformation. GFP fluorescence is shown as a heat map as indicated in A.
Scale bars: 500 µm (A); 50 µm (B); 300 µm (D).
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panels) in the inner ear suggesting that NF-κB signaling is strongly
activated in response to local infection.
Quantification of NF-κB activity in live animals
One advantage of the Tg(8×Hs.NFκB:GFP,Luciferase) fish
compared to previously available zebrafish NF-κB-responsive
reporters is the option for quantification of NF-κB activity by
bioluminescence. We measured bioluminescence in vivo with a
previously published method that is non-invasive and is easily
scalable (Lahiri et al., 2014). Briefly, living zebrafish larvae were
assayed in 96-multiwell plates by adding beetle luciferin potassium
salt solution directly to the water. In the first 4 dpf, concomitant with
the broadening of the GFP expression pattern (Fig. 4), luciferase
expression increased ∼10-fold (Fig. 7A). Treatment with JSH-23, a
drug that has been previously used to inhibit NF-κB activity in
zebrafish embryos (He et al., 2015), significantly reduced luciferase
levels at 2 dpf (Fig. 7B). Next, we measured the luciferase activity in
iκbαamorphants. Morpholino injection resulted in a significant
increase in luciferase activity in 2 dpf embryos compared to both
uninjected and control-injected embryos (Fig. 7C). These
experiments confirmed that the palindromic sequence drives
expression of both GFP and luciferase genes, and that the
bioluminescence can be used to quantify NF-κB activity in the
living fish.
The pSGNluc reporter does not interfere with cytokine
expression
Both in vitro and in vivo data confirmed that pSGNluc is a sensitive
reporter for monitoring NF-κB activation, likely due to the high
binding affinity of NF-κB proteins for the κB site sequence used in
this reporter. One could assume that the strong binding affinity of
pSGNluc reporter sites might interfere with the expression of
endogenous genes by outcompeting the transcription factor
complex, especially in NF-κB target genes that exhibit weak
transcriptional activity. To test this possibility in vitro,we
performed a competition assay by transfecting HEK293T cells
with different amounts of NF-κB-responsive plasmids, and
measuring interleukin-8 (IL-8) production and luciferase activity
24 h after stimulation with 10 ng/ml TNFα. We observed a positive
correlation between the amount of transfected NF-κB-responsive
plasmids and luciferase activity (Fig. S4A). However, increasing
amounts of transfected NF-κB-responsive plasmids did not
negatively affect IL-8 secretion (Fig. S4B). Surprisingly, even a
trend towards higher IL-8 secretion was visible. We next tested
whether cytokine expression was negatively affected in the
Tg(8xHs.NFκB:GFP,Luciferase) fish. We compared mRNA
levels of cytokine genes in transgenic and wild-type siblings
before and after inflammatory stimuli. We used UV irradiation to
induce inflammation in zebrafish embryos (Banerjee and Leptin,
2014), since NF-κB signaling plays a major role in the induction of
several cytokines during the inflammatory responses to this
stimulus. As expected, Tg(8×Hs.NFκB:GFP,Luciferase) embryos
exposed to UV light showed an increase of NF-κB activity
(Fig. 8A). We used quantitative real-time RT-PCR (qPCR) to
compare the mRNA level of selected cytokines in transgenic and
wild-type siblings (Fig. 8B). The cytokine genes interleukin-10
(il10), il12b and tnfa are direct target genes of NF-κB (Banerjee and
Leptin, 2014; Kennedy et al., 1997; Rivas and Ullrich, 1992).
Consistent with a previous study (Banerjee and Leptin, 2014), il10
and tnfa, but not il12b, were significantly upregulated in UV-treated
embryos (Fig. 8B). The results from qPCR showed that the
cytokines were expressed at comparable levels in the transgenic and
wild-type siblings. Taken together, these data suggest that the
pSGNluc reporter does not significantly interfere with the NF-κB
response in cell culture and zebrafish embryos.
DISCUSSION
We have established a new NF-κB-responsive reporter system.
Compared to other NF-κB-driven luciferase reporters, pSGNluc has
three major advantages. First, pSGNluc is inducible in response to
Fig. 6. In vivo visualization of NF-κB activity upon local wounding and
infection. (A) Time-lapse images derived from Movie 2 show NF-κB activation
after tailfin cut. GFP fluorescence in the top panel is shown as a heat map.
GFP is upregulated locally near the wound. (B,C) Injection of HcRed-
expressing E. coli into the notochord (B) or inner ear (C) of Tg(8×Hs.NFκB:
GFP,Luciferase) elicits NF-κB activation 20 h after infection. The bacterial
infection results in the accumulation of immune cells in the inner ear. GFP was
not expressed in the notochord or inner ear of uninfected control zebrafish.
The white dashed line in (C) demarcates the inner ear. Anterior is to the left.
Scale bars: 150 µm (A,C); 200 µm (B).
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TOOLS AND TECHNIQUES Journal of Cell Science (2017) 130, 648-657 doi:10.1242/jcs.196485
Journal of Cell Science

inflammatory stimuli, such as TNFα, at levels comparable to other
available reporters. Second, the reporter is significantly more
sensitive to both the proinflammatory cytokine TNFαand the
Nod1-specific inflammatory activator Tri-DAP than other
commonly used NF-κB reporters. This is likely because the
sequence and copy number of the κB site enhance the sensitivity of
the NF-κB-responsive reporter (Matsuda et al., 2007). Third, the bi-
directional property of the NF-κB-responsive promoter enables
simultaneous expression of two reporter genes: GFP expression
shows the kinetics and spatial aspects of a response in individual
cells in real time, while the luciferase assay allows a quantitative
evaluation of the global NF-κB activation throughout all stimulated
cells or tissues. This dual reporter system allows simultaneous
qualitative and quantitative monitoring of NF-κB activity in
response to inflammatory stimuli, the integration of which can
provide information beyond on–off activation. For example, the
broad GFP expression and high luciferase levels observed in cells in
culture after TNFαtreatment shows that the majority of transfected
cells responded to stimulation. In the case of Tri-DAP exposure, the
luciferase measurement suggested that NF-κB signaling was not
strongly induced after exposure; however, the mosaic GFP
expression after treatment showed that some cells do respond
strongly. It is not clear why only a subset of cells respond to Tri-
DAP, but we suspect it may be related to differences in drug uptake.
Tri-DAP enters epithelial cells via clathrin-mediated endocytosis,
then, it requires endosomal processing and an optimal pH for its
translocation into the cytosol (Sorbara and Philpott, 2011). This
may be an inefficient process, explaining why penetration of Tri-
DAP into the cytoplasm of HEK293T cells might not proceed
uniformly in all cells.
Two transgenic zebrafish lines carrying NF-κB-responsive
reporters have been generated previously (Banerjee and Leptin,
2014; Kanther et al., 2011). Both lines contained constructs with
multimerized κB sites to trigger GFP expression. The κB site
sequences used in these reporters are predicted to have lower
binding affinities for NF-κB proteins than those used in the
pSGNluc. One of these reporters (Banerjee and Leptin, 2014) does
not show detectable levels of GFP expression during embryonic
development and is less responsive to injury than the pSGNluc
reporter. The second reporter was used to study the effect of
microbiota on NF-κB activity in the intestine (Kanther et al., 2011).
This reporter drives GFP expression in the lateral line and intestine
during embryonic development, an activation pattern consistent
with our own observations for the pSGNluc reporter. However,
there are also some differences between the two lines, including the
dynamic expression in the olfactory neurons and stronger sporadic
expression in the epithelia, as well as the reduced expression in the
pharyngeal arches seen for the pSGNluc line. The GFP expression
seen with the pSGNluc reporter in the first 30 hpf is consistent
with previously described early expression of NF-κB transcription
factors (Correa et al., 2004) and reflected published spatiotemporal
expression patterns of genes involved in NF-κB signaling at these
stages, such as c-rel (Correa et al., 2004) and ikk1 (Correa et al.,
2005). This early NF-κB activation matches what is known
about the functional requirements for NF-κB signaling during
embryogenesis: during gastrulation NF-κB activation is involved in
coordinating the cell cycle and mesoendodermal cell movements
(Liu et al., 2009) and lack of NF-κB signaling in the notochord leads
to embryonic dorsalization and deformities (Correa et al., 2004).
The requirement of finely tuned NF-κB signaling during notochord
differentiation is also supported by the defects caused by high doses
of iκbαamorpholino.
The weak NF-κB activity in keratinocytes and its upregulation in
iκbαamorphants and in response to UV irradiation are also in
agreement with what is known about the role of NF-κB signaling in
keratinocyte inflammation and homeostasis (Wullaert et al., 2011).
The skin is exposed to multiple environmental stimuli, and NF-κB
signaling is required for the inflammatory response in this organ.
Additionally, NF-κB is a regulator of keratinocyte proliferation and
growth (Wullaert et al., 2011). The weak GFP signal in the skin is
Fig. 7. Quantification of NF-κB activity in live Tg(8×Hs.
NFκB:GFP,Luciferase) embryos. (A) Luciferase activity
during embryonic development. (B) Embryos at 1 dpf were
treated with 100 µM JSH-23, and luciferase activity was
measured at 2 dpf. Data represent the mean±s.d. luciferase
activity of three independent experiments conducted with
>12 embryos. (C) Transgenic embryos were injected with
0.3 mM standard control morpholino or iκbαamorpholino at
the one-cell stage and luciferase activity was measured at
2 dpf. Luciferase activity was increased in iκbαamorphants.
Each dot represents an individual embryo and the overall
mean±s.d. is shown. cps, counts per second.
Fig. 8. UV irradiation results in activation of the NF-κB pathway. (A) The induction was calculated by dividing luciferase values from embryos at 24 h post
UV irradiation by values of untreated transgenic (Tg) siblings at 2 dpf. The data represent the mean±s.d. induction of three independent experiments conducted
with >6 embryos. (B) Relative expression of il10,tnfa and il12b in transgenic (Tg) and wild-type (WT) siblings without and 24 h post UV irradiation. The β-actin
gene was used as a reference for normalization. Each dot represents an individual embryo and the overall mean±s.d. is shown.
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TOOLS AND TECHNIQUES Journal of Cell Science (2017) 130, 648-657 doi:10.1242/jcs.196485
Journal of Cell Science

also in agreement with findings in transgenic mice carrying an
NF-κB-responsive reporter (Carlsen et al., 2002). In mice, IκBαis
expressed in the skin and preventsthe nuclear localization of NF-κB
proteins. Newborn mice lacking IκBαexhibit increased NF-κB
activation in the skin, which triggers keratinocyte proliferation and
epidermal hyperplasia (Klement et al., 1996). A similar phenotype
was observed in RelA-knockout mice or after overexpression of a
dominant-negative mutant version of Iκbαin the epidermal
keratinocytes (Seitz et al., 2000; Zhang et al., 2004). The tissue-
specific response to iκbαaknockdown as well as to local
inflammation suggest that the GFP signal in the Tg(8×Hs.NFκB:
GFP,Luciferase) embryos recapitulates the activity of NF-κB
signaling in zebrafish embryos.
A novel aspect of our reporter line compared to the existing
NF-κB-responsive reporters (Banerjee and Leptin, 2014; Kanther
et al., 2011) is that it allows quantification of NF-κB activity by
bioluminescence measurement. We showed that induction of
luciferase in vivo correlated with the increase in NF-κB activity as
measured by GFP expression, and that it responded to the
knockdown of the NF-κB inhibitor iκbαain the same way as
GFP did. Our assays measure luciferase activity in each larva
individually in such a way that the larva is unharmed and can
survive for several days, allowing continuous tracking of reporter
activity (Lahiri et al., 2014). This represents a significant
improvement over a previously published method to measure NF-
κB activity in zebrafish with a luciferase reporter in which lysis of
the embryo was required for activity measurement (Alcaraz-Perez
et al., 2008). Bioluminescence imaging of NF-κB expression in a
luciferase transgenic mouse model has been used as a tool to screen
anti-NF-κB drug candidates (Robbins and Zhao, 2011). The
zebrafish is a model ideally suited for high-throughput, whole-
organism drug screening (Zon and Peterson, 2005) through the use
of fluorescence-based analysis both of reporter activity (Wang et al.,
2015) and bacterial load (Ordas et al., 2015; Veneman et al., 2013).
We therefore anticipate that the zebrafish transgenic pSGNluc will
become a valuable tool to study many aspects of NF-κB signaling
during development and inflammation in real time. The transgenic
reporter could also provide a cost-effective improvement for high-
throughput assays in drug discovery.
MATERIALS AND METHODS
DNA constructs
Multimerized NF-κB-binding sites with the idealized sequence 5′-GGGA-
ATTC CC- 3′separated by 6 bp spacers were generated by oligonucleotide
ligation. A fragment containing eight NF-κB-binding sites was inserted in the
pSGHluc plasmid (Bajoghli et al., 2004). The resulting plasmid ( pSGNluc)
contains eight NF-κB sites flanked by two 260 bp long minimal CMV
promoters. The bi-directional promoter drivesthe firefly luciferase gene with a
3′UTR of the globin gene and an SV40 polyA at one side, and GFP with a
bovine growth hormone (bGH) polyA at the other side. The sequence of the
pSGNluc plasmid has been deposited at NCBI under the accession number
KY129798. The β-galactosidase expression plasmid is described in Philpott
et al. (2000). pGL4.32 was purchased from Promega and IL-8 luciferase
reporter is described in Bowie et al. (2000). The original name of IgκB-Luci is
(IgK)3-conaluc and is described in Munoz et al. (1994).
Cell culture assays
In vitro analysis was performed as described previously (Zurek et al., 2011).
Briefly, cells of the human embryonic kidney cell line HEK293T were plated
at a density of 30,000 cells per well in a 96-multiwell plate. Transfection
mixes were prepared for triplicate assays. A total amount of 51 ng DNA with
13 ng of the respective NF-κB-responsive vector, 8.6 ng β-galactosidase- or
mCherry-expressing plasmid, and 29.4 ng pcDNA3.1 was used per well.
HEK293T cells were transfected with DNA mixes using X-tremeGENE
TM
9 DNA transfection reagent (Roche). Unless otherwise indicated,
transfected cells were directly stimulated with either TNFα(Invivogen) or
Tri-DAP (Invivogen) at the indicated concentrations, and measurements
were performed after 24 h. For kinetic measurements, cells were transfected,
incubated overnight and stimulated with TNFαat 10 ng/ml for 8, 6, 3, 2 or
1 h before cell lysis. For the measurement of luciferase and β-galactosidase
activities, cells were lysed at 24 h post transfection. Cells were lysed in
100 µl lysis buffer (25 mM Tris-HCl pH 8.0, 8 mM MgCl
2
, 1% Triton X-
100, 15% glycerol) per well. To measure the luciferase activity, 50 µl of the
cell lysates were transferred to a white non-transparent 96-multiwell plate
and luciferase activity was quantified as relative luminescence units (RLU)
in a multiplate reader (Enspire, Perkin Elmer) upon automatic adding of
100 µl luciferase substrate solution [lysis buffer containing 1.3 µM ATP and
770 ng/ml D-luciferin (Sigma)]. To measure β-galactosidase activity, the
remaining 50 µl cell lysate was supplemented with 100 µl of 1 mg/ml o-
nitrophenyl-β-D-galactopyranosid (ONPG) in 60 mM Na
2
HPO
4
,40mM
NaH
2
PO
4
, 10 mM KCl, 1 mM MgSO
4
at pH 7.0 per well and incubated at
37°C for 30 min. Then, absorption was measured in a photometer at 405 nm
(620 nm reference). β-Galactosidase activity was used to normalize
luciferase activity (nRLU) in each well. Three independent experiments,
each performed in triplicate, were conducted for each assay.
ELISA
To determine IL-8 concentrations, supernatants of HEK293T cells without
and with TNFα(10 ng/ml) stimulation were analyzed with the human
CXCL8/IL8 DuoSet ELISA kit (R&D Systems) according to the
manufacturer’s protocol. The IL-8 concentration in unstimulated cells was
below the level of detection.
Zebrafish
All animal experiments described in the present study were conducted on
embryos younger than 5 dpf under the rules of the European Molecular
Biology Laboratory and the guidelines of the European Commission,
Directive 2010/63/EU. The zebrafish strain used in this study was Danio
rerio wild-type TL. The stable transgenic line Tg(krt19:dTomato) was
described previously (Fischer et al., 2014). To develop the stable transgenic
zebrafish line, pSGNluc plasmid was co-injected with I-SceI meganuclease
enzyme in 1× I-SceI buffer (New England, BioLabs) into the blastomere at
the one-cell stage as described previously (Aghaallaei et al., 2007). Three
zebrafish founders were identified with similar GFP expression patterns.
One founder was crossed with the wild-type TL strain and their progeny
was used for this work. The transgenic zebrafish carrying the pSGNluc
is named Tg(8xHs.NFκB:GFP,Luciferase)
hdb5
in accordance with the
Zebrafish Nomenclature Guidelines (https://wiki.zfin.org/display/general/
ZFIN+Zebrafish+Nomenclature+Guidelines) and was approved by the
Zebrafish Nomenclature Committee.
Morpholino and bacterial microinjections
Antisense morpholino (MO) for zebrafish ikbαa(5′-TGCGGCTCTGTG-
TAAATCCATGTTC-3′; He et al., 2015) and standard control morpholino
were obtained from Gene Tools. They were prepared as 1 mM and 3 mM
stock solutions in double-distilled H
2
O, respectively. Morpholino at a
concentration of 0.3 mM or 1 mM together with 100 mM KCl was injected
into the blastomere of zebrafish embryos at the one-cell stage. Non-
pathogenic Gram-negative Escherichia coli BL21 expressing HcRed
(Aghaallaei et al., 2010) were microinjected into the transgenic pSGNluc
embryos at 3 dpf as described previously (Nguyen-Chi et al., 2014).
UV treatments
UV treatment in zebrafish embryos was performed as described previously
(Banerjee and Leptin, 2014). Briefly, embryos at 24 hpf (n=50) were sorted
into a petri dish and exposed to 24 mJ/cm
2
of UV. Embryos were maintained
in E3 medium at 28°C for 24 h prior to total RNA extraction or luciferase
measurement.
Drug treatment
JSH-23 (Selleckchem Inc) was diluted to a stock concentration of 10 mM in
DMSO, aliquoted and stored at −80°C. For treatment, the drug was added
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TOOLS AND TECHNIQUES Journal of Cell Science (2017) 130, 648-657 doi:10.1242/jcs.196485
Journal of Cell Science

directly to the E3 medium of manually-dechorionated 1 dpf embryos to a
final concentration of 100 µM. Luciferase activity was measured 24 h after
drug exposure.
Flow cytometry and fluorescence microscopy
For FACS analysis, HEK293T cells were plated in 24-well plates, directly
transfected with pSGNluc and pmCherry plasmids in a 1:1 ratio, and
stimulated with TNFαor Tri-DAP at the indicated concentrations. 24 h later,
cells were trypsinized, resuspended in 5% fetal calf serum (FCS) in PBS and
analyzed with the FACSCanto system (BD Biosciences). To image mCherry
and GFP expression in vitro, HEK293T cells were plated on glass
coverslips, transfected and stimulated as above. After 24 h, stimulated
cells were fixed with 4% paraformaldehyde in PBS for 20 min. After a short
wash in 1×PBS, fixed cells were mounted with Mowiol4.88 (Sigma)
supplemented with bis-benzimide to stain the nuclei. For live-cell imaging,
HEK293T cells were plated in compartmentalised glass-bottom petri dishes
(Greiner bio-one) before transfection and stimulation. Images were taken on
a Zeiss Axiovert 200 M equipped with a Plan-Apochromat 20×0.8 NA M27
objective and AxioCamMR3 camera (Carl Zeiss, Jena) or a Leica DMi8
equipped with a FluotarL 20×0.4 NA objective (Leica) and Orca Flash
4.0LT camera (Hamamatsu).
To image GFP expression in vivo, transgenic pSGNluc zebrafish embryos
were anesthetized with tricaine methanesulfonate and mounted in 1.5% low-
melting-point agarose. In vivo imaging of the embryos was performed with a
Zeiss Lightsheet Z.1 and Zeiss LSM 780 NLO 2-Photon confocal
microscopes. To visualize NF-κB activation upon infection or injury,
time-lapse experiments were carried out with an Ultraview ERS spinning
disk (PerkinElmer) confocal microscope using a 40× water-immersion
objective. Images were analyzed with Imaris software as described
previously (Bajoghli et al., 2015).
Luciferase measurement in live embryos
Single embryos were transferred into individual wells of a 96-multiwell
plate (Nunc) in 100 µl E3 medium (without methylene blue), supplemented
with 0.5 mM beetle luciferin potassium salt solution (Promega), and the
plate was sealed using an adhesive Top Seal sheet (Packard).
Bioluminescence from each embryo was then assayed at room
temperature using a Top-count NXT scintillation counter (2-detector
model; Packard).
Whole-mount in situ hybridization
RNA in situ hybridization of wild-type zebrafish embryos was performed as
described previously (Bajoghli et al., 2009) using digoxigenin-labeled RNA
antisense probe for iκbαa(accession number, BC068382; nucleotides 230–
903) and iκbαb(accession number, BC050175; nucleotides 242–689).
Quantitative RT-PCR analysis
Total RNA was extracted from pools of 25 embryos using TRIzol (Life
Technologies) following the manufacturer’s protocol. 1 µg RNA samples
were treated with 1 µl RQ1 RNase-Free DNase (Promega) before first-strand
cDNA synthesis with random hexamer primers and Superscript III Reverse
Transcriptase (Thermo Fisher). The first-strand cDNA was directly used as a
template in PCR reactions. qPCR was carried out using the SYBR Green kit
(Applied Biosystems) on the ABI 7500 Real-Time PCR System. The data
were analyzed in Microsoft Excel using the ΔCt method with β-actin as a
reference gene for normalization. All primer sequences used in this study
were described previously (Banerjee and Leptin, 2014).
Statistical analysis
Prism software (version 6, GraphPad Software Inc.) was used for graphing
and statistical analysis. Unpaired, two-tailed Student t-tests were used to
compare the means of different data sets.
Acknowledgements
We thank the Advanced Light Microscopy Facility (AMLF) at EMBL for continuous
support and PerkinElmer and Zeiss for support of the AMLF. We are grateful to
Nicholas S. Foulkes (Institute of Toxicology and Genetics, Karlsruhe, Germany) for
providing the experimental materials for luciferase measurement in live zebrafish
embryos, Stephen A Renshaw (University of Sheffield, UK) for providing the Tg
(NFκB:EGFP) fish and to Matthias Hammerschmidt (University of Cologne,
Germany) for the Tg(krt19:dTomato) fish. We thank Francesca Peri for hosting our
zebrafish, Sinja Kraus and Omnia El Said Ibrahim for experimental help; Yvonne
Postma for technical support; Marvin Albert for help with the processing of SPIM
data; Joanna Natalia Buffoni and Cornelia Henkel for the care of zebrafish. Wethank
Sanjita Banerjee for help in the generation of the zebrafish Tg(8×Hs.NFκB:GFP,
Luciferase) line.
Competing interests
The authors declare no competing or financial interests.
Author contributions
P.K. established the zebrafish pSGNluc transgenic line, performed in vivo analyses,
contributed to the design of the work and co-wrote the manuscript. K.E. performed
the reporter assays in human cells. T.A.K. conducted initial studies, provided
scientific discussion and supervised the in vitro characterization work in human
cells. M.L. provided scientific discussion, contributed to critical revision for important
intellectual content and final approval. B.B. designed and developed the pSGNluc
reporter, conducted initial studies, planned and oversaw the work, interpreted the
data, designed figures, co-wrote the paper and all authors read and edited the
manuscript.
Funding
The laboratory of M.L. is supported by the European Molecular Biology Organization
(EMBO) and European Molecular Biology Laboratory (EMBL), and grants and
fellowships to B.B. (European Commission, EMBL EU Marie Curie Action Cofund),
P.K. (European Commission Marie-Curie Initial Training Network FishForPharma;
FP7-PEOPLE-2011-ITN, grant PITN-GA-2011-289209). Preparatory work was
funded by the Deutsche Forschungsgemeinschaft (DFG) SFB 670 ‘Zellautonome
Immunita
̈t’. The laboratory of T.A.K. is supported by the Deutsche
Forschungsgemeinschaft (KU1945/4-1).
Data availability
The sequence of the pSGNluc plasmid has been deposited at NCBI under the
accession number KY129798 (https://www.ncbi.nlm.nih.gov/nuccore/KY129798)
and the the Tg(8×Hsa.NFκB:GFP,Luciferase) line described here has been
deposited in the European Zebrafish Resource Center (EZRC) for public distribution
(in the ZFIN network under https://zfin.org/ZDB-TGCONSTRCT-161209-5).
Supplementary information
Supplementary information available online at
http://jcs.biologists.org/lookup/doi/10.1242/jcs.196485.supplemental
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TOOLS AND TECHNIQUES Journal of Cell Science (2017) 130, 648-657 doi:10.1242/jcs.196485
Journal of Cell Science

Supplementary Information
Fig. S1. Comparison between Tg(8xHs.NFκB:GFP,Luciferase) and two other
previously developed zebrafish NF-κB-responsive transgenic lines. (A)
Tg(NFκB:EGFP) embryos display stronger GFP signal in the skin compared
to the Tg(8xHs.NFκB:GFP,Luciferase) embryos at 3dpf. Note the
Tg(NFκB:EGFP) line was originally described in Kanther et al., 2011. (B)
Comparison between Tg(8xHs.NFκB:GFP,Luciferase) and Tg(NFκB-EGFP)
after tailfin cut. The GFP was examined 40 hours post-injury (hpi). Note equal
laser intensity was used to illustrate that Tg(NFκB-EGFP) embryos display
weaker GFP signal in the injured site compared to the
Tg(8xHs.NFκB:GFP,Luciferase) embryos. Yellow arrows indicate cells with
GFP expression. Note Tg(NFκB-EGFP) line was originally described in
Banerjee and Leptin, 2014. Anterior is to the left. Scale bars: 200µm (A) and
30µm (B).
DICGFP
low high
GFP intensity
Tg(NFκB:EGFP)
A B
Tg(8xHs.NFκB:GFP,Luciferase)
Tg(NFκB-EGFP)
Tg(8xHs.NFκB:GFP,Luciferase)
40 hpi 40 hpi
(Kanther et al., 2011) (Banerjee and Leptin, 2014)
J. Cell Sci. 130: doi:10.1242/jcs.196485: Supplementary information
Journal of Cell Science • Supplementary information

Fig. S2. NF-κB activity in the skin of zebrafish embryos at 4dpf. Trunk region
of a Tg(8xHs.NFκB:GFP,Luciferase [green]; krt19:dTomato [red]) embryo at
4dpf illustrating the sporadic NF-κB activity in the basal keratinocytes
(arrowheads). Note krt19 is mainly expressed in the basal keratinocyte layer
(Fischer et al., 2014). Yellow arrow indicates GFP expression in the
proctodeum. White arrows indicate GFP expression in cells surrounding the
lateral line. Anterior is to the left. Scale bars: 100µm (top panel) and 30µm
(bottom panel).
krt19:dTomato merge
Tg(8xHs.NFκB:GFP,Luciferase)
J. Cell Sci. 130: doi:10.1242/jcs.196485: Supplementary information
Journal of Cell Science • Supplementary information

Fig. S3. Expression pattern of iκbαa and iκbαb in zebrafish embryos. Whole-
mount RNA in situ hybridization of iκbαa (A-D) and iκbαb (E-H) in zebrafish
embryos at 2-3dpf. A-C, E-G show lateral views. Anterior is to the right. D and
H show the head of embryos from the dorsal side. Black arrows indicate
expression in the proctodeum. Red arrows indicate expression in the lateral
line. Black arrowheads indicate iκbαa expression in the nasal vesicle.
ikbαaikbαb
48hfp72hfp
A E
B F
C GD H
J. Cell Sci. 130: doi:10.1242/jcs.196485: Supplementary information
Journal of Cell Science • Supplementary information

Fig. S4. NF-κB-responsive reporter does not interfere with the IL-8 production.
HEK293T cells were transfected with distinct amounts of the indicated
plasmids. The nRLU level (A) and IL-8 production (B) were measured 24
hours after stimulation with TNFα (10ng/ml). Note the basal IL-8 level was
below the detection limit in all unstimulated cells (data not shown). Values
represent mean + SEM.
IgκB-Luci
pGL4.32
pSGNluc
1
2
4
8
16
32
64
128
256
IL-8 (p g/ml)
B
2
25
2
20
2
15
2
10
2
5
nRLU
0110100 0110100 0110100
Plasmid (ng )
0110100 0110100 0110100
Plasmid (ng )
A
J. Cell Sci. 130: doi:10.1242/jcs.196485: Supplementary information
Journal of Cell Science • Supplementary information

Table S1. The luciferase activity (nRLU) of NF-κB-responsive reporters
before and 24 hours after stimulation with TNFα at 10ng/µl
Reporter
nRLU (mean ± SEM)
N*
Unstimulated
After stimulation
IL-8-Luc
21552±13464
7784063±299357
5
IgκB-Luci
7352±3520
6317149±2679460
5
pGL4.32
13943±5732
5213250±2299480
4
pSGNluc
137059±45013
58877451±2527590
5
*N, number of independent experiments conducted in triplicates.
J. Cell Sci. 130: doi:10.1242/jcs.196485: Supplementary information
Journal of Cell Science • Supplementary information

Table S2. The luciferase activity (nRLU) of NF-κB-responsive
reporters 24 hours after stimulation with Tri-DAP at 10µg/ml
Reporter
nRLU (mean ± SEM)*
IL-8-Luc
41578±937
IgκB-Luci
170925±11448
pGL4.32
167266±23920
pSGNluc
2277672±143324
*Data from 3 independent experiments conducted in triplicates.
J. Cell Sci. 130: doi:10.1242/jcs.196485: Supplementary information
Journal of Cell Science • Supplementary information

A high-sensitivity, bi-directional reporter to monitor NF-κB activity in cell culture and
zebrafish in real-time
Movies:
Movie S1. Time-lapse in vitro imaging of pSGNluc transfected cells without
simulation (left panel), treated with Tri-DAP at 10µg/ml (middle panel) or
TNFα at 5ng/ml (right panel). Times are in hours. Scale bar: 100µm
J. Cell Sci. 130: doi:10.1242/jcs.196485: Supplementary information
Journal of Cell Science • Supplementary information

Movie S2. Time-lapse in vivo imaging of Tg(8xHs.NFκB:GFP,Luciferase)
embryos after tailfin cut. GFP fluorescence is shown as false-colour heat map.
Anterior is to the left. Times are in hours. Scale bar: 70µm
J. Cell Sci. 130: doi:10.1242/jcs.196485: Supplementary information
Journal of Cell Science • Supplementary information