Advancing Pathway Analysis With Custom Luciferase Reporters
Matthew Robers, Brock Binkowski, Natasha Karassina, Pete Stecha, Jey Cheng, Jennifer Wilkinson, Aileen Paguio, Chris Eggers, Braedy Butler, Frank Fan, and Mei Cong
Promega Corporation; 2800 Wood Hollow Road, Madison WI 53711
1. Abstract
[email protected]
7. Protein Stability Regulates Key Cytokine and Stress
Response Pathways
4. Cytokine and Pathway Analysis for Biologics
To enable investigation of key cellular signaling pathways, Promega has
developed a portfolio of bioluminescent reporter gene assays using Firefly
and Renilla luciferases. In combination with best-in-class luciferase
detection reagents, these genetic reporter systems enable interrogation of
important cellular responses involved in cancer, inflammation, and CNS
disease. To address specialized customer needs in our industrial and
research markets, Promega has a new custom assay service team
dedicated to applying these enabling technologies through strategic
external research collaborations. The performance of this technology
portfolio is presented, including novel applications of luciferase reporters to
interrogation of cytokine, stress, and toxicity pathway responses.
Latest Research Materials: Vectors and Cell Lines for
Cytokines and Pathways
JAK/STAT1 INF-
Dose Response to IFN Treatment
SLAS 2012
Target Protein
T1/2 Normal
((min))
T1/2 Induced (min)
Inducer
HIF1
50
200-250
Hypoxia/mimetics
IB
100
5
TNFa/other inflammatory cytokines
p53
20
300-400
Genotoxic stress (UV/chemical DNA damage,
etc)
Nrf2
Oxidative Stress
100
Fold Induction
80
Cytokine Pathway Response Element
60
40
20
0
0.0001
0.01
1
100
10000
[IFN-], ng/mL
Name
GLi
ISRE
GAS
SIE
DBE
C/EBP
LEF/TCF
SMAD3/4
Hedgehog
JAK/STAT1/2 INF-
JAK/STAT1 INF-
JAK/STAT3 IL-6
PI3K/AKT
Multiple
Wnt
TGF-beta
pGL4[luc2P/Gli-RE/Hygro] Vector
pGL4[luc2P/ISRE/Hygro] Vector
pGL4[luc2P/GAS-RE/Hygro] Vector
pGL4[luc2P/SIE-RE/Hygro] Vector
pGL4[luc2P/DBE-RE/Hygro] Vector
pGL4[luc2P/C/EBP-RE/Hygro] Vector
pGL4[luc2P/LEF/TCF-RE/Hygro] Vector
pGL4[luc2P/SMAD3/4-RE/Hygro] Vector
10-15
30-40
-Catenin
<60
>200
Wnt
FOXO
>300
<60
Growth Factors
PDCD4
300
<60
Insulin / PI3K
JAK/STAT3 IL6
c-Jun
Activator/pathway
Fold response
e
400
EC50 = 3.7 ng/ml
g
300
200
100
Reporter Construct
Cell Background/marker
INF-STAT1:STAT1
GAS-luc2P
INF-STAT1:STAT1
GAS-luc2P
HepG2/Hygro
IL-6/STAT3:STAT3
SIE-luc2P
HEK293/Hygro
-2
0
2
IL-6/STAT3:STAT3
SIE-luc2P
HepG2/Hygro
>200
Stress
20
300-400
Stress
<60
>300
LiCl
HEK293/Hygro
8. Protein Stability Reporters Using Renilla Luciferase Enable
Signaling Pathway Measurements in Real-time
0
-4
<60
c-Myc
c/EBP
4
log IL-6 [ng/ml]
For complete offering, visit www.promega.com/LRM
Light is proportional to Protein X concentration dynamics
2. Toxicity Pathways: A Common Pathway Architecture
Luciferin
Protein X-Luciferase
5. CASE STUDY #1: Detecting CNTFR Using SIE-Luc2P/HepG2
Stable Cell Line
Latest Research Materials: Vectors and Cell
Lines for Stress and Toxicity
Light
O2
Protein Stability
Sensor
Constitutive
Promoter
Luciferase
Gene X
Figure
g
2
Figure 1
pGL4.27[Xbp1-luc2P/minP/Hygro] Vector
CNTF alone
pGL4.27[ATF4-luc2P/minP/Hygro] Vector
10000
Heavy metal stress
MRE
pGL4.27[MTF1-luc2P/minP/Hygro] Vector
Heat shock
HSE
pGL4.27[HSE-luc2P/minP/Hygro] Vector
Hypoxia
HIF1
pGL4.27[HIF1-luc2P/minP/Hygro] Vector
MAPK
AP1
pGL4.27[AP1-luc2P/minP/Hygro] Vector
AhR
pGL4.27[XRE-luc2P/minP/Hygro] Vector
Myc/Max
pGL4.27[Mycluc2P/minP/Hygro] Vector
Xenobiotic stress
CNTF + 200ng/ml CNTFRa
5000
-4
-2
0
2
CNTF + 200ng/ml CNTFRa
5
-4
4
3.0
-2
0
2
4
log [CNTF], ng/ml
log [CNTF], ng/ml
1496
1.5
1.0
0
For complete offering,
visit www.promega.com/LRM
Cell Line Background
ARE
HEK293
Hypoxia
HIF1
HEK293
Hypoxia
HIF1
HepG2
MAPK
AP1
HEK293
*Ras/MEK-1
SRE
HEK293
*RhoA (G12/13)
SRF
HEK293
2
3
1.5
1.0
0.8
0.6
0.4
2
4
6
8
90
80
70
RLuc-ODD Stability Assay
HRE-luc2P GloResponse
60
50
40
30
20
10
0
0.2
-10
10 - 2
0.0
0
4
100
100 ng/mL TNF
Vehicle
1.0
0
10
1
2
3
10 - 1
4
10 0
10 1
10 2
[Phenanthroline], uM
time (h)
Time(h)
RLuc-ODD Stability Assay HRE-luc2P GloResponse
7.667
6.000
EC50
EnduRen™ offers benefit of assay normalization (to RLU at time=0)
154.2
Materials and Methods: Ciliary neurotrophic factor (CNTF) is a 23 kDa neurocytokine which is expressed in both the peripheral and central
nervous system and provide hopes for its possible clinical application in the treatment of human neurodegenerative diseases. Like other
cytokines, CNTF exerts its biological effects through the activation of a multichain receptor complex consisting of a ligand-specific subunit
(CNTFR), gpl30 and the LIF receptor-(LIFR-). GloResponse™ SIE-Luc2P reporter is stably expressed in HepG2 cells where LIFR is
endogenously expressed with no CNTFR detection. Stable cells were plated in assay media (MEM + 10% FBS) at 40K cells/80 ul/well in 96 well
plate and incubated overnight at 37°C with 5% CO2. Next day,10ul of 10X soluble CNTFR was added to appropriate wells (or media to
untreated wells) with subsequent 10ul of 10X CNTF dose addition. Cells were incubated 6 hours prior to One-Glo substrate (Promega, Cat.#
E6110) addition. As shown in the data, in the present of exogenously added soluble CNTFRLIF receptor bearing HepG2 cells respond to
CNTF at lower doses, consistent to the published data.
2.0
time (h)
Hypoxia Sensor
Genotoxic Stress Sensor
p53-RLuc Stabilization
8 Hour Timepoint
RLuc-ODD Stabilization
3 hour timepoint
2.5
2.0
1.5
1.0
0.5
10 - 1
10 0
10 1
[Phenanthroline], uM
10 2
0.8
2.5
2.0
1.5
1.0
10 - 2
TNF sensor
10 - 1
10 0
10 1
[Etoposide], uM
10 2
Comparison of NFkB RE vs IkB degradation
in HEK293 Cells
IkB-RLuc Degradation
30 minute timepoint
3.0
3.0
Normalized Response
(normalized to RLU at time=0)
Unstressed Cell
Response Element
Antioxidant
1
110
1.2
100 uM Etoposide
Vehicle
2.5
0.5
0.5
CNTF +
200ng/mL CNTFRa
CNTF alone
2.0
TNF sensor
IkB-RLuc Stabilization
Timecourse
3.0
50 uM Phenanthroline
Vehicle
2.5
TF
Toxicity Pathway
Genotoxic Stress Sensor
p53-RLuc Stabilization
Timecourse
RLuc-ODD Stabilization
Timecourse
CNTF alone
10
0
0
Ec50, ng/mL
+
Hypoxia Sensor
15
Normalized Response
pGL4.27[ATF6-luc2P/minP/Hygro] Vector
Xbp1
Myc
Degradation
Ub
ATF6
Comparison of HRE to HIF/ODD Stabilization
in HEK293 Cells
Normalized Response
TF
ATF4
Normalized Response
(normalized to RLU at time=0)
TF
Ub Ub
Ub Ub
ER stress
ER stress
ER stress
20
15000
Normalized Response
(normalized to RLU at time=0)
Sensor
pGL4.27[p53-luc2P/minP/Hygro] Vector
Normalized Response
(normalized to RLU at time=0)
Sensor
TF
pGL4.27[NRF2-luc2P/minP/Hygro] Vector
p53
Normalized Response
(normalized to RLU at time=0)
Sensor
SIE-luc2P HepG2
SIE-luc2P HepG2
Name
ARE
Normalized Response
(normalized to RLU at time=0)
Transduction
Response Element
DNA damage/p53
Fold response
Toxicity Pathway
Antioxidant
RLU
Stressor
10 3
0.7
0.6
0.5
0.4
110
100
90
80
70
60
50
40
30
20
10
0
-10
10 - 2
10 - 1
10 0
10 1
10 2
10 3
[TNF], ng/mL
0.3
0.2
10 - 2
IkB-Rluc Stability
NFkB-luc2P GloResponse
10 - 1
10 0
10 1
10 2
10 3
EC50
IkB-Rluc Stability NFkB-luc2P GloResponse
4.861
2.451
[TNF], ng/mL
6. CASE STUDY #2: Leveraging SRE-Luc2P/HEK293 Reporter Cell
y Development
p
Line to Bioassay
3. Superior Functional Performance in Transient Transfection
Heat Shock
XRE-luc2P
HepG2 Transient Transfection
HSE-luc2P
HeLa Transient Transfection
310 0 5
HRE-luc2P
HepG2 Stable Cell Line
8.010
20000
15000
110 0 5
10000
510 0 4
10 - 2
10 - 1
10 0
10 1
10 2
10 3
-10
-9
-8
-7
-6
-5
0
10 - 1 0
-4
log [TCDD], M
[PMA], nM
10 - 9
10 - 8
10 - 7
10 - 6
0
10 - 2
10 - 5
10 - 1
10 0
10 1
MEK-1
MAPK
10 2
10
-9
10
-8
10
-7
10
-6
DNA Damage/p53
Metal Stress
p53-luc2P
U2OS Transient Transfection
ARE-luc2P
HEK293 Stable Pool
10
200000
15
410 0 4
10
210 0 4
10 - 5
10 - 4
10 - 3
2.510 5
5
110
10 - 6
5.010 5
310 0 4
0
-5
[tBHQ], M
-4
-3
-2
-1
0
log [Mytomycin], µg/ml
EC50
24 hours
0.4596
1
2
04
0
-12
-10
-8
-6
log [ZnSO4], M
-4
-2
0
-4
-3
-2
-1
0
log [ICG-001], µM
1
2
CArG
Luc
10 - 1 0
10 - 9
4000
3000
2000
1000
0
-10
10 - 8
10 - 7
-9
-8
-7
-6
log[tunicamycin] (M)
-5
9. Conclusions
[Growth Factor], g/ml
Materials and Methods: GloResponse™ SRE-Luc2P HEK293 cells were plated in 96 well plate overnight. Cells were
stimulated with serially-diluted agonist and allowed to incubate approximately 18 hours. For detection of luciferase activity
after stimulation, to 100uL of culture medium, an equal volume of ONE-Glo™ detection reagent was added to each well. After
10 minutes of equilibration, luminescence was measured on a GloMax™ Multi plate reader set to 0.5s of luminescence
integration time.
5000
normalized RLU
400000
0
10 - 1 1
SRF
S
SRE= Serum Response Element-luc2P (destabilized Fluc)
ATF4-Luc2P
Hela Transient Transfection
6 hours
24 hours
RLU
600000
RLU
Fold response
RLU
800000
0
10 - 7
7.510 5
Ets
ER Stress ATF4
Myc‐Luc2P
HCT116 Transient Transfeection
Metal Stress-luc2P
HeLa Transient Transfection
510 0 4
400000
[Tunicamycin], M
Myc/Max
610 0 4
20
1000000
EGF
FGF
NGF
VEGF
600000
Materials and Methods: Measurement of inducible stabilization of various Renilla luciferase fusion proteins. Figure 1: HEK293 cells were
transfected with pF5A DNA encoding humanized Renilla luciferase (Rluc) fusion proteins using Fugene HD and seeded in 96-well format. Left
panels; cells were transfected with DNA encoding the ODD domain of HIF1a fused to the c-terminus of Rluc. To achieve appropriate expression
levels of Rluc-ODD, reporter DNA was diluted into pGem3Z DNA to yield 50pg/well. Middle panels; cells were transfected with DNA encoding p53
fused to the N-terminus of Rluc at 0.5ng/well as described above. Right panels; cells were transfected with DNA encoding IkB fused to the Nterminus of Rluc at 50pg/well as described above. 24 hours post-transfection, cell medium was replaced with CO2-independent medium
supplemented with 20uM EnduRen™ Reagent, followed by equilibration for 4 hours at 37C. Cells were then stimulated with the serially diluted
agonist indicated, for the time indicated. Luminescence was then measured in real-time on a GLOMAX Multi+ Plate reader set to 5 minute
measurement intervals. For data normalization, RLUs of each sample were normalized to the RLU values immediately after stimulation for that
sample. Figure 2: Comparable pharmacology between luciferase fusion and reporter gene assay for measuring hypoxia response (top) and TNF
response (bottom)..
-5
SRF
S
Antioxidant
800000
Comparable pharmacology between luciferase
fusion and reporter gene assay
200000
0
10 - 1 0
[Phenanthroline], uM
[17-AAG] (M)
1000000
P
6.010 5
2.010 5
5000
0
-11
5
4.010 5
10000
2.010 6
0
10 - 3
P
Ras
Raf
1.010 6
210 0 5
Profiling Endogenous Pathways in HEK293 Cells
1.210 6
20000
RLU
RLU
RLU
ATF6 RE-luc2P
HeLa Cells Transient Transfection
30000
25000
210 0 5
4.010 6
RTK
ER Stress ATF6
RLU
AP-1-luc2P
HEK293 Transient Transfection
6.010 6
Hypoxia
RLU
Xenobiotic Stress
RLU
MAPK
Live cell readout enables data normalization, with excellent Z’ (0.6-0.8)
-4
www.promega.com
 Promega now offers fully validated Firefly luciferase reporter vectors for key toxicity and
cytokine signaling pathways
 Modular solutions exist for the investigation of endogenous and exogenous receptor
biology using GloResponse™ stable cell lines
 Novel pathway analysis tools are available using luciferase fusion technologies
 Promega’s Custom Assay Service (CAS) team provides expertise in generation of custom
vectors or stable cell lines encoding bioluminescent reporters