Cignal Reporter Assays (45 Pathways)

1
Cignal Reporter Assays (45 Pathways)
Pathway Transcription Factor MAPK/ERK Elk-1/SRF
MAPK/JNK AP-1 MEF2  MEF2  c-myc Myc/Max
Nanog Nanog NF?B NF?B  Notch RBP-Jk Oct4 
Oct4  Pax6 Pax6 PI3K/AKT  FOXO  PKC/Ca
NFAT PPAR  PPAR  Progesterone PR Retinoic
Acid RAR Retinoid X RXR Sox2 Sox2 SP1  SP1 
STAT3  STAT3  TGFß SMAD2/3/4 Vitamin
D Vitamin D Receptor  Wnt TCF/LEF Xenobiotic
AhR
Pathway Transcription Factor Amino 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 GR Heat Shock Response  HSF 
Heavy Metal Stress  MTF1  Hedgehog GLI HNF4
HNF4 Hypoxia HIF-1 Interferon
Regulation IRF-1 Type I Interferon STAT1/STAT2
Interferon Gamma STAT1/STAT1
KLF4 KLF4 Liver X LXR
http//www.sabiosciences.com/cellassay.php
2
Cignal Reporter Assays
Cell-Based Assays for Rapidly Analyzing Pathway
Signaling Activity 
Samuel J. Rulli, Jr., Ph.D. qPCR and Pathway
Signaling Application Scientist
3
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

4
Post-Genomics Research Challenge

• Moving from a Signaling Flowchart Perspective
• to a Signaling Network Perspective.

Basic Signal Transduction Flowchart
5
Post-Genomics Research Challenge
Signal Transduction Cascade A B C
D (40 proteins)
6
Cell-based Assay Research Challenges

• Variability within experiments
• Well-to-well and plate-to-plate reproducibility
  are notoriously challenging with cell-based
  assays.
• Variability between experiments
• Reliable results often depend upon the status
  of the cells, and who in the lab is conducting
  the experiments.
• Assay Performance
• Sensitivity, specificity, signal to noise ratio,
  and simplicity are of utmost importance.

7
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

8
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

9
Cignal Pathway Reporter System Products
10
Cignal Reporter Assays (45 Pathways)
Pathway Transcription Factor MAPK/ERK Elk-1/SRF
MAPK/JNK AP-1 MEF2  MEF2  c-myc Myc/Max
Nanog Nanog NF?B NF?B  Notch RBP-Jk Oct4 
Oct4  Pax6 Pax6 PI3K/AKT  FOXO  PKC/Ca
NFAT PPAR  PPAR  Progesterone PR Retinoic
Acid RAR Retinoid X RXR Sox2 Sox2 SP1  SP1 
STAT3  STAT3  TGFß SMAD2/3/4 Vitamin
D Vitamin D Receptor  Wnt TCF/LEF Xenobiotic
AhR
Pathway Transcription Factor Amino 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 GR Heat Shock Response  HSF 
Heavy Metal Stress  MTF1  Hedgehog GLI HNF4
HNF4 Hypoxia HIF-1 Interferon
Regulation IRF-1 Type I Interferon STAT1/STAT2
Interferon Gamma STAT1/STAT1
KLF4 KLF4 Liver X LXR
11
Cignal Lenti Assays (35 Pathways)
Pathway Transcription Factor Amino 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
GR Heat Shock Response  HSF  Heavy Metal
Stress  MTF1  Hedgehog Gli HNF4 HNF4 Hypoxia
HIF-1 Interferon Regulation IRF-1 Type I
Interferon STAT1/STAT2 Interferon Gamma
STAT1/STAT1 KLF4 KLF4 Liver X LXR
Pathway Transcription Factor MAPK/ERK Elk-1/SRF
MAPK/JNK AP-1 MEF2  MEF2  c-myc Myc/Max
Nanog Nanog NF?B NF?B  Notch RBP-Jk Oct4 
Oct4  Pax6 Pax6 PI3K/AKT  FOXO  PKC/Ca
NFAT PPAR  PPAR  Progesterone PR Retinoic
Acid RAR Retinoid X RXR Sox2 Sox2 SP1  SP1 
STAT3  STAT3  TGFß SMAD2/3/4 VDRE 
Vitamin D Receptor  Wnt TCF/LEF Xenobiotic A
hR
12
REPORTER DESIGN Dual-Luciferase Assays
The Transcriptional Regulatory Elements (TREs)
are what establish the pathway specificity of
each reporter!
13
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

14
REPORTER DESIGN Dual-Luciferase Assays
The Transcriptional Regulatory Elements (TREs)
are what establish the pathway specificity of
each reporter!
15
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/treatme
  nt
• 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

16
Dual Luciferase Format Minimizes Variability
Dual-Luciferase Assays NFkB Reporter Assay
Single-Luciferase Assays NFkB Reporter Assay
CV0.07
CV0.70
CV0.09
CV0.69
NFkB Reporter NFkB siRNA
NFkB Reporter Neg. control siRNA
17
REPORTER DESIGN Dual-Luciferase Assays
The Transcriptional Regulatory Elements (TREs)
are what establish the pathway specificity of
each reporter!
Mammalian codon-optimized AND Destabilized
firefly luciferase
18
Engineered Luciferase and the SignalNoise Ratio
Modified Luciferase Background NFkB Reporter
Signal (no treatment post-transfection)
Modified Luciferase NFkB Reporter
Signal Following TNF? Induction
Negative Control
NFkB Reporter Stable Luc
NFkB Reporter Destabilized Luc
Negative Control
NFkB Reporter Stable Luc
NFkB Reporter Destabilized Luc
19
REPORTER DESIGN Dual-Luciferase Assays
The Transcriptional Regulatory Elements (TREs)
are what establish the pathway specificity of
each reporter!
20
Cignal TRE Maximize Luciferase Signal
Dual-Luciferase Assays Utilizing Cignal p53 TRE
p53 Reporter DMSO
p53 Reporter 1µM Doxorubicin
21
Cignal Reporter Assays How It Works
22
Cignal Reporter Assays (GFP)
Pathway Transcription Factor Amino 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
GR Heat Shock Response  HSF  Heavy Metal
Stress  MTF1  Hedgehog Gli HNF4 HNF4 Hypoxia
HIF-1 Interferon Regulation IRF-1 Type I
Interferon STAT1/STAT2 Interferon Gamma
STAT1/STAT1 KLF4 KLF4 Liver X LXR
Pathway Transcription Factor MAPK/ERK Elk-1/SRF
MAPK/JNK AP-1 MEF2  MEF2  c-myc Myc/Max
Nanog Nanog NF?B NF?B  Notch RBP-Jk Oct4 
Oct4  Pax6 Pax6 PI3K/AKT  FOXO  PKC/Ca
NFAT PPAR  PPAR  Progesterone PR Retinoic
Acid RAR Retinoid X RXR Sox2 Sox2 SP1  SP1 
STAT3  STAT3  TGFß SMAD2/3/4 VDRE 
Vitamin D Receptor  Wnt TCF/LEF Xenobiotic A
hR
23
GFP-based Reporter Small Molecule Studies
Fluorometry
Fluorescent Microscopy
Treatment of Cignal SRE (GFP) transfectants with
10 serum and 10 ng/ml PMA activates the MAPK/ERK
signaling pathway.
24
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)
25
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

26
Application Study
p53 Pathway Interaction Study

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

27
Post-Genomic Challenges Pathway Interactions
28
Constructing Signaling Networks
29
Application Study
p53 Pathway Interaction Study

• Question
• What is impact of p53 gene expression knock down
  on
• the p53 signaling pathway AND other
  cancer-relevant
• signaling pathways?
• Approach
• Carry out a p53 RNA interference experiment with
• multiple cancer-relevant Cignal Reporter Assays.

30
Cignal Finder Cancer Array p53 RNAi Study

• p53 Cignal Reporter
• RBP-Jk Cignal Reporter
• TCF/LEF Cignal Reporter
• E2F Cignal Reporter
• AP1 Cignal Reporter
• SRE Cignal Reporter
• SMAD Cignal Reporter
• 8. NFkB Cignal Reporter
• 9. Myc Cignal Reporter

2
4
1
3
5
8
11
12
9
10
6
7
31
Results Cancer Array Application Study
Impact of p53 siRNA Treatment
32
Post-Genomics Challenge Pathway Interactions
33
Summary Cignal Reporter Assay System

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