sample & assay technologies - 1 - cignal reporter assays (45 pathways) pathwaytranscription...

Sample & Assay Technologies- 1 -

Cignal Reporter Assays (45 Pathways)

Pathway Transcription FactorAmino Acid Deprivation ATF4/ATF3/ATF2 Androgen Androgen Receptor Antioxidant Response Nrf2 & Nrf1 ATF6 ATF6 C/EBP C/EBP cAMP/PKA CREB Cell Cycle E2F/DP1 DNA Damage p53 EGR1 EGR1 ER Stress CBF/NF-Y/YY1 Estrogen ER GATA GATA Glucocorticoid GRHeat Shock Response HSF Heavy Metal Stress MTF1 Hedgehog GLIHNF4 HNF4Hypoxia HIF-1 Interferon Regulation IRF-1Type I Interferon STAT1/STAT2 Interferon Gamma STAT1/STAT1 KLF4 KLF4Liver X LXR

Pathway Transcription FactorMAPK/ERK Elk-1/SRF MAPK/JNK AP-1 MEF2 MEF2 c-myc Myc/Max Nanog NanogNFκB NFκB Notch RBP-Jk Oct4 Oct4 Pax6 Pax6PI3K/AKT FOXO PKC/Ca++ NFAT PPAR PPAR Progesterone PRRetinoic Acid RAR Retinoid X RXRSox2 Sox2SP1 SP1 STAT3 STAT3 TGFβ SMAD2/3/4 Vitamin D Vitamin D Receptor Wnt TCF/LEFXenobiotic AhR

http://www.sabiosciences.com/cellassay.php


Cignal™ Reporter Assays

Samuel J. Rulli, Jr., Ph.D. qPCR and Pathway Signaling Application Scientist

Cell-Based Assays for Rapidly Analyzing Pathway Signaling Activity


Topics to be Covered

Utility of the Cignal Reporter Assays• Challenges Associated with Cell-Based Assays• Solutions Cignal Reporter Assays Provide

.How Cignal Reporter Assays Work• Cignal Pathway Reporters Product Family• Dual-Luciferase Assays• Luciferase Engineered for High Performance• Optimized Transcriptional Regulatory Elements (TREs)

.Application Study: p53 Pathways


Post-Genomics Research Challenge:

Moving from a Signaling Flowchart Perspective

to a Signaling Network Perspective.

DNA damage

ATM/ATR

p53

Cell cycle arrest/Apoptosis

Basic SignalTransductionFlowchart


Post-Genomics Research Challenge:

p38

HIF1α

Ub

Ub

14-3-3

MDM2

P P

P

P

E2F

c-FosP

Rb

P

P

BRCA1

AngiogenesisInhibitionAngiogenesisInhibition

Cell CycleProgressionCell Cycle

Progression

Gene ExpressionGene Expression

Cell SurvivalCell Survival

DNA DamageDNA Damage

Hypoxia InducedHIF1 ExpressionHypoxia InducedHIF1 Expression

ApoptosisApoptosis

p53Degradation

p53Degradation

DNA RepairDNA Repair

Ac

AcCAK

MDM2

PIAS1

JNK

Caspase

BCL2

BAI1

TSP1

GADD45

RbCDK2

CyclinD1

CDK4

CyclinE E2F

PCAF

DNA-PK

PTEN

ATM

p53

Akt

p53

p53

Ub

ATR

Sirt

HDAC

c-Abl

p53PML

ChemotherapyChemotherapyIonizing

RadiationIonizing

RadiationHypoxiaHypoxia p53 SignalingUVUV

p21CIP

PTEN

ProteasomeProteasome

HIPK2 CSNK1

p53

GSK3β

Bax

DR5

F

a

s

Ac

p53

p300

Chk2

Chk1

Signal TransductionCascadeA B C D

(~40 proteins)


Cell-based Assay Research Challenges

.Variability within experimentsWell-to-well and plate-to-plate reproducibility are notoriously

challenging with cell-based assays.

.Variability between experimentsReliable results often depend upon the “status” of the cells, and

who in the lab is conducting the experiments.

.Assay PerformanceSensitivity, specificity, signal to noise ratio, and simplicity are of

utmost importance.


What Cignal Reporter Assays Provide

Functionally validated dual-reporter formulation Minimizes experimental variability, increasing the biological relevance

Ready-to-use reporters and controls, in transfection ready format: Enables simple and rapid analysis of the regulation of 45 signal transduction pathways

Custom-engineered transcriptional regulatory elements (TREs) and destabilized, codon-optimized firefly luciferase Increases the signal to noise ratio and maximizes assay sensitivity and specificity


Cignal Pathway Reporter System Products

Easy totransfect cell lines

Primary cells, stem cells,and difficult to transfect cell lines

EndpointAssays

EndpointAssays

Dynamic Live Cell Assays

Dynamic Live Cell Assays

Cignal Dual-Luciferase Reporter Assays

Cignal Lenti Luciferase Reporters

Cignal GFP Reporter Assays

Cignal Lenti GFP Reporters

Single PathwayAssays




10-PathwayArrays

10-PathwayArrays


Cignal Reporter Assays (45 Pathways)

Pathway Transcription FactorAmino Acid Deprivation ATF4/ATF3/ATF2 Androgen Androgen Receptor Antioxidant Response Nrf2 & Nrf1 ATF6 ATF6 C/EBP C/EBP cAMP/PKA CREB Cell Cycle E2F/DP1 DNA Damage p53 EGR1 EGR1 ER Stress CBF/NF-Y/YY1 Estrogen ER GATA GATA Glucocorticoid GRHeat Shock Response HSF Heavy Metal Stress MTF1 Hedgehog GLIHNF4 HNF4Hypoxia HIF-1 Interferon Regulation IRF-1Type I Interferon STAT1/STAT2 Interferon Gamma STAT1/STAT1 KLF4 KLF4Liver X LXR

Pathway Transcription FactorMAPK/ERK Elk-1/SRF MAPK/JNK AP-1 MEF2 MEF2 c-myc Myc/Max Nanog NanogNFκB NFκB Notch RBP-Jk Oct4 Oct4 Pax6 Pax6PI3K/AKT FOXO PKC/Ca++ NFAT PPAR PPAR Progesterone PRRetinoic Acid RAR Retinoid X RXRSox2 Sox2SP1 SP1 STAT3 STAT3 TGFβ SMAD2/3/4 Vitamin D Vitamin D Receptor Wnt TCF/LEFXenobiotic AhR


Cignal Lenti Assays (35 Pathways)

Pathway Transcription FactorAmino Acid Deprivation ATF4/ATF3/ATF2 Androgen Androgen Receptor Antioxidant Response Nrf2 & Nrf1 ATF6 ATF6 C/EBP C/EBP cAMP/PKA CREB Cell Cycle E2F/DP1 DNA Damage p53 EGR1 EGR1 ER Stress CBF/NF-Y/YY1 Estrogen ER GATA GATA Glucocorticoid GRHeat Shock Response HSF Heavy Metal Stress MTF1 Hedgehog GliHNF4 HNF4Hypoxia HIF-1 Interferon Regulation IRF-1Type I Interferon STAT1/STAT2 Interferon Gamma STAT1/STAT1 KLF4 KLF4Liver X LXR

Pathway Transcription FactorMAPK/ERK Elk-1/SRF MAPK/JNK AP-1 MEF2 MEF2 c-myc Myc/Max Nanog NanogNFκB NFκB Notch RBP-Jk Oct4 Oct4 Pax6 Pax6PI3K/AKT FOXO PKC/Ca++ NFAT PPAR PPAR Progesterone PRRetinoic Acid RAR Retinoid X RXRSox2 Sox2SP1 SP1 STAT3 STAT3 TGFβ SMAD2/3/4 VDRE Vitamin D Receptor Wnt TCF/LEFXenobiotic AhR


REPORTER DESIGN: Dual-Luciferase Assays

Reporter Construct Firefly luciferaseTATA boxTRE

Renilla luciferaseTATA boxCMVNormalization Construct

Pathway-targeted transcriptional regulatory elements (45)

Constitutive transcriptional regulatory element (1)

The Transcriptional Regulatory Elements (TREs) are what establish the

pathway specificity of each reporter!


Using Cignal Reporters: What You Need

• Cignal Reporter Assay

• Reliable transfection reagent and protocol (Attractene, HiPerFect)

• Test Biological•shRNA (SureSilencing shRNA)• expression vectors (QIAgenes)• protein/peptide• small molecule

• Detection Instrument and Luciferase Substrates






Constitutive transcriptional regulatory element (1)




Dual Luciferase Assays: Why Is It Important?

Sources of Variability in Cell-based Assays:

• Number of cells seeded per well• Transfection efficiencies within and across plates• Multichannel pipettor inconsistencies• “Edge effects” influencing cell culture• Viability of cells following transfection/treatment• Lysis efficiencies within and across plates• Variability inherent to reporter assay

• Changes due to the designed treatments of cells• RNA interference• Overexpression• Recombinant protein/peptide/growth factor• Small molecule

Dual-Luciferase Assaydesign corrects for these unwanted sources of variability.


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Dual Luciferase Format Minimizes Variability

Dual-Luciferase Assays:NFkB Reporter Assay

CV=0.09

CV=0.07

Single-Luciferase Assays:NFkB Reporter Assay

CV=0.69

CV=0.70

NFkB Reporter+ NFkB siRNA

NFkB Reporter+ Neg. control siRNA

NFkB Reporter+ NFkB siRNA

NFkB Reporter+ Neg. control siRNA





Mammalian codon-optimized AND

Destabilized firefly luciferase




Engineered Luciferase and the Signal:Noise Ratio

Modified Luciferase:Background NFkB Reporter Signal

(no treatment post-transfection)

Modified Luciferase:NFkB Reporter Signal

Following TNF Induction

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100

150

200

250

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NegativeControl

NFkB Reporter:Stable Luc

NFkB Reporter:Destabilized Luc

NegativeControl

NFkB Reporter:Stable Luc

NFkB Reporter:Destabilized Luc


Cignal TRE Maximize Luciferase Signal

Dual-Luciferase AssaysUtilizing Cignal p53 TRE

0

20

40

60

80

100

120

140

160

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p53 Reporter+ DMSO

p53 Reporter+ 1µM Doxorubicin

Dual-Luciferase AssaysUtilizing Common p53 TRE

p53 Reporter+ DMSO

p53 Reporter+ 1µM Doxorubicin

125-Fold2.5-Fold


Cignal Reporter Assays: How It Works


Cignal Reporter Assays (GFP)

Pathway Transcription FactorAmino Acid Deprivation ATF4/ATF3/ATF2 Androgen Androgen Receptor Antioxidant Response Nrf2 & Nrf1 ATF6 ATF6 C/EBP C/EBP cAMP/PKA CREB Cell Cycle E2F/DP1 DNA Damage p53 EGR1 EGR1 ER Stress CBF/NF-Y/YY1 Estrogen ER GATA GATA Glucocorticoid GRHeat Shock Response HSF Heavy Metal Stress MTF1 Hedgehog GliHNF4 HNF4Hypoxia HIF-1 Interferon Regulation IRF-1Type I Interferon STAT1/STAT2 Interferon Gamma STAT1/STAT1 KLF4 KLF4Liver X LXR

Pathway Transcription FactorMAPK/ERK Elk-1/SRF MAPK/JNK AP-1 MEF2 MEF2 c-myc Myc/Max Nanog NanogNFκB NFκB Notch RBP-Jk Oct4 Oct4 Pax6 Pax6PI3K/AKT FOXO PKC/Ca++ NFAT PPAR PPAR Progesterone PRRetinoic Acid RAR Retinoid X RXRSox2 Sox2SP1 SP1 STAT3 STAT3 TGFβ SMAD2/3/4 VDRE Vitamin D Receptor Wnt TCF/LEFXenobiotic AhR


GFP-based Reporter: Small Molecule Studies

Treatment of Cignal SRE (GFP) transfectants with 10% serum and 10 ng/ml PMAactivates the MAPK/ERK signaling pathway.

FluorometryFluorescent Microscopy


Cignal Reporter Assays: Two Modalities

Dual-luciferase Format - Quantitative endpoint luminescence assay - Exceptional reproducibility, sensitivity, and signal-to-noise ratio - Average expression from a population of cells

GFP Format - Dynamic live cell assay - Single cell resolution - Readout flexibility

(flow cytometry, fluorescent microscopy, fluorometry)


Application Study

Question:What is impact of p53 gene expression knock down on

the p53 signaling pathway AND other cancer-relevant

signaling pathways?

p53 Pathway Interaction Study


Post-Genomic Challenges: Pathway Interactions

Wnt Signaling NFkB Signaling

E2F Signaling

MAPK/ERK Signaling

Myc Signaling

MAPK/JNK Signaling

TGFβ Signaling

Notch Signaling

?

?

?

?

?

?

?

?

?Hypoxia Signaling

p53 Signaling


Constructing Signaling Networks


Application Study

Question:What is impact of p53 gene expression knock down on the p53 signaling pathway AND other cancer-relevantsignaling pathways?

Approach:Carry out a p53 RNA interference experiment with multiple cancer-relevant Cignal Reporter Assays.

p53 Pathway Interaction Study


Cignal Finder Cancer Array : p53 RNAi Study

1 2 3 4 5 6 7 8 11

1. p53 Cignal Reporter

2. RBP-Jk Cignal Reporter

3. TCF/LEF Cignal Reporter

4. E2F Cignal Reporter

5. AP1 Cignal Reporter

6. SRE Cignal Reporter

7. SMAD Cignal Reporter

8. NFkB Cignal Reporter

9. Myc Cignal Reporter

10. HIF Cignal Reporter

11. Cignal Negative Control

12. Cignal Positive Control

129 10

p53 siRNA

Neg. control siRNA


Results: Cancer Array Application Study

Impact of p53 siRNA Treatment


Post-Genomics Challenge: Pathway Interactions

MAPK/ERK Signaling

Notch Signaling

Hypoxia Signaling

p53 Signaling


Summary: Cignal Reporter Assay System

.Breadth of Pathways and Research Applications• 45 Pathway-Focused Reporter Assays available individually or as biologically focused panels.

.PerformanceExceptional Sensitivity, Specificity, Signal-to-Noise Ratio, and Reproducibility.

Ready-to-use, validated, preformatted transient assays.

.Multiple Reporters:•Dual-luciferase quantitative endpoint assays.•GFP dynamic live cell assays with single cell resolution.

http://www.sabiosciences.com/cellassay.php

sample & assay technologies - 1 - cignal reporter assays (45 pathways) pathwaytranscription...

Documents

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degradation of p53

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nonphosphorylated p53

transactivation domain

p53mediated function