Introduction

A common stress response pathway architecture can

be measured using transcription or protein stability

Conclusions

www.promega.com

NanoLuc® and GloSensor™ Technologies Enabling Kinetic Analysis of Signaling Pathways in iCell® Cardiomyocytes Jennifer Wilkinson, Jeff Kelly, Richard Somberg, Chris Eggers, Brock Binkowski, Jim Hartnett, Frank Fan, Keith Wood, Mei Cong, and Matthew Robers Promega Corporation; 2800 Wood Hollow Road, Madison WI 53711

TF

TF

TF

Degradation

+ TF

Sensor

Unstressed Cell

Stressor

Sensor

Transduction

Sensor

Target

Protein

T1/2 Normal

(min)

T1/2 Induced

(min)

Inducer

HIF1a 50 200-250 Hypoxia/mimetics

IkBa 100 5 TNFα/other inflammatory cytokines

p53 20 300-400 Genotoxic stress (UV/chemical DNA damage,

etc)

Nrf2 10-15 30-40 Oxidative Stress

b-Catenin <60 >200 Wnt

FOXO >300 <60 Growth Factors

PDCD4 300 <60 Insulin / PI3K

c-Jun <60 >200 Stress

c-Myc 20 300-400 Stress

c/EBP <60 >300 LiCl

Although traditional reporter gene assays have proven powerful tools to interrogate cell signaling pathways, transcriptional responses generally occur at distal signaling endpoints, limiting their suitability for real-time pathway analysis. Consequently, alternate methods that measure signaling responses at upstream pathway nodes are desirable as complements to transcriptional reporters. Promega has applied a new set of ultrasensitive bioluminescent reporter technologies ideally suited for these applications. First, NanoLuc® technology is ideally suited as a protein fusion tag for measuring changes in protein lifetime following induction of stress response signaling. Via genetic attachment of NanoLuc to various transcription factors, induction of ROS and hypoxia responses can be measured. GloSensor™ technology exploits analyte-sensitive forms of permuted firefly luciferase for the purposes of quantifying intracellular cAMP levels upon induction of G-protein coupled receptors. The sensitivity of these reporters make them well-suited for applications in iCell® Cardiomyocytes using simple transfection methods.

Materials and Methods. iCell® Cardiomyocytes were seeded into 96-well plates. 3-5 days post-

seeding, cells were transfected using either pGL4 ARE-luc2P, HRE-luc2P, or NanoLuc® fusion

constructs (diluted 1:100 into pGEM®-3Z promoterless DNA). 24h post-transfection, cells were

stimulated for either 6 hours (for the RE reporter) or 3 hours (for the stability sensor). Following

induction of ROS or hypoxia, either ONE-Glo™ Luciferase Assay System or Nano-Glo™ Luciferase

Assay Reagents were added at 1:1 volumes. Luminescence was then quantified on a GloMax®-

Multi+ plate reader.

• Protein lifetime is an excellent surrogate readout for stress

response signaling

• NanoLuc is an excellent protein fusion partner for protein lifetime

measurements

• GloSensor™ technology enables real-time analysis of receptor

signaling in iCell® Cardiomyocytes

• These bioluminescent sensor assays also enable a non-destructive

endpoint for kinetic analysis of signaling pathways in iCell®

Cardiomyocytes

Description of the figure. Many stress response pathways utilize a common pathway

architecture. In unperturbed cells, levels of transcription factors are regulated by the

ubiquitin-proteasome system. Upon pathway induction, the stability of these proteins

dramatically changes, resulting in activation of transcription. Lower Panel. Many key

signaling pathways can be measured using response element-based reporter gene

assays.

Use of NanoLuc® Luciferase as a Small, Bright Protein

Fusion Reporter for Protein Lifetime Measurements

Exploiting Protein Lifetime as a Readout of

Stress Response Signaling in iCell® Cardiomyocytes Live Cell, Non-Destructive Format for Monitoring

Hypoxia Response in iCell® Cardiomyocytes

Real-time Analysis of Adrenergic Signaling using

GloSensor™ cAMP in iCell® Cardiomyocytes

A n tio x id a n t R e s p o n s e E le m e n t

(A R E -L u c 2 P )

lo g [D , L S u lfo ra p h a n e ], u M

fold

re

sp

on

se

-2 -1 0 1 2

0

5

1 0

1 5

2 0

A n tio x id a n t R e s p o n s e E le m e n t

(A R E -lu c 2 P )

lo g [ tB H Q ], u M

fold

re

sp

on

se

-3 -2 -1 0 1 2

0

1 0

2 0

3 0

N rf2 -N a n o L u c S ta b il ity S e n s o r A s s a y

lo g [D ,L S u lfo ra p h a n e ], u M

fold

re

sp

on

se

-2 -1 0 1 2

0

5

1 0

1 5

2 0

N rf2 -N a n o L u c S ta b il ity S e n s o r A s s a y

lo g [ tB H Q ], u M

fold

re

sp

on

se

-4 -2 0 2

0

5

1 0

1 5

2 0

H y p o x ia R e s p o n s e E le m e n t

(H R E -lu c 2 P )

lo g [ IO X 2 ], u M

RL

U

-4 -2 0 2

0

1 0 0 0 0

2 0 0 0 0

3 0 0 0 0

H IF 1 a -N a n o L u c S ta b ility S e n s o r

lo g [C o C l2 ] , u M

RL

U

-2 0 2

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

H y p o x ia R e s p o n s e E le m e n t

(H R E -lu c 2 P )

lo g [M L 2 2 8 ], u M

RL

U

-6 -4 -2 0

0

5 0 0 0 0

1 0 0 0 0 0

1 5 0 0 0 0

H y p o x ia R e s p o n s e E le m e n t

(H R E -lu c 2 P )

lo g [C o C l2 ] , u M

RL

U

-4 -2 0 2 4

0

5 0 0 0 0

1 0 0 0 0 0

1 5 0 0 0 0

H y p o x ia R e s p o n s e E le m e n t

(H R E -lu c 2 P )

lo g [P h e n a n th ro lin e ] , u M

RL

U

-4 -2 0 2

0

5 0 0 0 0

1 0 0 0 0 0

1 5 0 0 0 0

2 0 0 0 0 0

H IF 1 a -N a n o L u c S ta b ility S e n s o r

lo g [ IO X 2 ], u M

RL

U

-4 -2 0 2

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

H IF 1 a -N a n o L u c S ta b ility S e n s o r

lo g [P h e n a n th ro lin e ] , u M

RL

U

-4 -2 0 2

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

H IF 1 a -N a n o L u c S ta b ility S e n s o r

lo g [M L 2 2 8 ], u M

RL

U

-5 -4 -3 -2 -1 0

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

TF TF

TF

TF

Sensor

Sensor

T im e c o u s re o f Is o p r o te r e n o l S t im u la tio n

G lo S e n s o r c A M P 2 2 F

0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

0 n M IS O

0 .6 4 n M IS O

3 .2 n M IS O

1 6 n M IS O

8 0 n M IS O

4 0 0 n M IS O

2 u M IS O

1 0 u M IS O

T im e (s e c )

Fo

ld

Re

sp

on

se

D o s e R e s p o n s e o f Is o p r o te r e n o l S tim u la t io n

G lo S e n s o r c A M P 2 2 F

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

-9 -8 -7 -6 -5 -4

2 m in

1 0 m in

6 0 m in

lo g [c o m p o u n d ] (M )

Fo

ld

Re

sp

on

se

T im e c o u s re o f F o rs k o lin S tim u la tio n

G lo S e n s o r c A M P 2 2 F

0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0

0 .1

1

1 0

1 0 0

1 0 0 0

0 n M F S K

1 2 .8 n M F S K

6 4 n M F S K

3 2 0 n M F S K

1 .6 u M F S K

8 u M F S K

4 0 u M F S K

2 0 0 u M F S K

T im e (s e c )

Fo

ld

Re

sp

on

se

D o s e R e s p o n s e o f F o rs k o lin S tim u la t io n

G lo S e n s o r c A M P 2 2 F

0

1 0 0

2 0 0

3 0 0

-9 -8 -7 -6 -5 -4

lo g [F o rs k o lin ] (M )

Fo

ld

Re

sp

on

se

Diagram of Glosensor™ cAMP Activation. The assay is based on the GloSensor™ Technology, a genetically

modified form of firefly luciferase which has been modified with a cAMP-binding protein moiety inserted into

unique N and C termini. Upon binding of cAMP, conformational change is induced leading to increased

luciferase activity.

• Live Cell Assay: Excels at kinetic and modulation studies of Gas-coupled receptors signaling through cAMP.

• Transient or Stable Expression: GloSensor™ cAMP Assay is utilized by transiently expressing a receptor of

interest and the biosensor in the cell line of choice. Alternatively, stably transfected cell lines with both the

biosensor and the receptor of interest can be made.

• Simple Protocol: Cells are pre-equilibrated with GloSensor™ cAMP Reagent , then cells are treated with

specific agonists/antagonists or compounds, and luminescence is measured in real-time (typically 10-30

minutes).

N

C

New N- &

C-termini

Fuse wt N- &

C-termini Analyte (cAMP) binding domain

Protein X-Luciferase Substrate

O2

Light

Protein Stability

Sensor

Constitutive

Promoter Luciferase Gene X

Light is proportional to Protein X concentration dynamics

H IF 1 a -N a n o L u c S ta b ility S e n s o r

L iv e c e ll a s s a y , N o n -D e s tru c tiv e E n d p o n t

2 h t im e p o in t

[c o m p o u n d ], u M

No

rm

ali

ze

d R

es

po

ns

e

(no

rm

ali

ze

d t

o R

LU

at

tim

e=

0)

1 0 -4 1 0 -2 1 0 0 1 0 2 1 0 4

0

5

1 0

1 5

2 0 P h e n h a n th ro lin e

C o C l2

H IF 1 a -N a n o L u c S ta b ility S e n s o r

L iv e c e ll a s s a y , N o n -D e s tru c tiv e E n d p o n t

1 h t im e p o in t

[c o m p o u n d ], u M

No

rm

ali

ze

d R

es

po

ns

e

(no

rm

ali

ze

d t

o R

LU

at

tim

e=

0)

1 0 -4 1 0 -2 1 0 0 1 0 2 1 0 4

0

5

1 0

1 5

2 0 P h e n h a n th ro lin e

C o C l2

Furimazine substrate

NanoLuc (NLuc) is an ATP-independent luciferase that utilizes a novel coelenterazine analog

(furimazine) to produce a high intensity, glow-like luminescent signal. The enzyme, evolved from a

deep-sea shrimp, is much brighter than firefly luciferase (FLuc) and provides superior sensitivity as a

genetic reporter. Unlike other forms of luciferase, NLuc is ideally suited for both standard (lytic) and

secretion based (non-lytic) reporter gene applications. The small size of the gene and encoded

protein enable viral applications and protein fusions, respectively. The enhanced thermal stability is

expected to reduce the number of false hits in primary high-throughput screening (HTS).

log[luciferase] (pM)

Lu

min

escen

ce (

RL

U)

-3 -2 -1 0 1 2 3 4 5 6 7102

103

104

105

106

107

108

109

101 0 Firefly (FLuc)

Renilla (RLuc)

NLuc

RLU comparison using purified protein

NLuc: 100-fold increased

sensitivity

ROS Responses in iCell® Cardiomyocytes, Measured by

Reporter Gene Assays and NanoLuc® Protein Stability Sensors

Hypoxia Responses in iCell® Cardiomyocytes, Measured by

Reporter Gene Assays and NanoLuc® Protein Stability Sensors

Materials and Methods. iCell® Cardiomyocytes were seeded into 96-well plates. 3-5 days post-

seeding, cells were transfected HIF1a-NanoLuc® fusion construct (diluted 1:100 into pGEM®-3Z

promoterless DNA). 24h post-transfection, cell medium was replaced with Opti-MEM®

supplemented with Furimazine. Luminescence was measured prior to stimulation, and then at 1h

and 2h post-stimulation. Data were normalized to pre-stimulation RLUs.

Materials and Methods; iCell® Cardiomyocytes were transfected with the pGloSensor-22F cAMP

plasmid, followed by overnight at 37C + 0.5% CO2. Prior to stimulation, media on the plate was

exchanged with 90 uL/well of CO2-Independent Medium + 2% (v/v) GloSensor™ cAMP Reagent

(Promega Cat.# E1290) and incubated one hour at 28C to allow equilibration with the substrate. The

plate was placed into a Varioskan Flash luminometer set at 28C and read in kinetic mode for 30 minutes

with 0.5 second integration to allow temperature equilibration and determine the basal GloSensor™

signal for each well. At this point, the stimulant was added. The plate was again read in kinetic mode for

over an hour. To calculate the fold response of the GloSensor, the RLUs for each well were divided by

the average of the last three pre-reads immediately before compound addition.

Response Element Readout Protein Stability Sensor

Luc2P NLuc NLuc