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Cignal Reporter Assays (45 Pathways)

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1 Cignal Reporter Assays (45 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 NFκB NFκB  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  TGFβ SMAD2/3/4 Vitamin D Vitamin D Receptor  Wnt TCF/LEF Xenobiotic AhR

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 Utility of the Cignal Reporter Assays
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. DNA damage ATM/ATR p53 Cell cycle arrest/Apoptosis 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 Utility of the Cignal Reporter Assays
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
Easy to transfect cell lines Primary cells, stem cells, and difficult to transfect cell lines Endpoint Assays Dynamic Live Cell Assays Cignal Dual-Luciferase Reporter Assays Cignal Lenti Luciferase Reporters Cignal GFP GFP Reporters Single Pathway 10-Pathway Arrays

10 Cignal Reporter Assays (45 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 NFκB NFκB  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  TGFβ SMAD2/3/4 Vitamin D Vitamin D Receptor  Wnt TCF/LEF Xenobiotic AhR

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 NFκB NFκB  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  TGFβ SMAD2/3/4 VDRE  Vitamin D Receptor  Wnt TCF/LEF Xenobiotic AhR

12 REPORTER DESIGN: Dual-Luciferase Assays
The Transcriptional Regulatory Elements (TREs) are what establish the pathway specificity of each reporter! Pathway-targeted transcriptional regulatory elements (45) Reporter Construct Firefly luciferase TATA box TRE Renilla luciferase TATA box CMV Normalization Construct Constitutive transcriptional regulatory element (1)

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! Pathway-targeted transcriptional regulatory elements (45) Reporter Construct Firefly luciferase TATA box TRE Renilla luciferase TATA box CMV Normalization Construct Constitutive transcriptional regulatory element (1)

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/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 Assay design corrects for these unwanted sources of variability.

16 Dual Luciferase Format Minimizes Variability
Single-Luciferase Assays: NFkB Reporter Assay Dual-Luciferase Assays: NFkB Reporter Assay CV=0.07 CV=0.70 CV=0.09 CV=0.69 NFkB Reporter + NFkB siRNA NFkB Reporter + Neg. control siRNA NFkB Reporter + NFkB siRNA + 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 Reporter Construct Firefly luciferase TATA box TRE Renilla luciferase TATA box CMV Normalization Construct

18 Engineered Luciferase and the Signal:Noise Ratio
Modified Luciferase: NFkB Reporter Signal Following TNF Induction Modified Luciferase: Background NFkB Reporter Signal (no treatment post-transfection) WHY? 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! Pathway-targeted transcriptional regulatory elements (45) Reporter Construct Firefly luciferase TATA box TRE Renilla luciferase TATA box CMV Normalization Construct

20 Cignal TRE Maximize Luciferase Signal
Dual-Luciferase Assays Utilizing Common p53 TRE p53 Reporter + DMSO + 1µM Doxorubicin Dual-Luciferase Assays Utilizing Cignal p53 TRE 125-Fold 2.5-Fold 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 NFκB NFκB  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  TGFβ SMAD2/3/4 VDRE  Vitamin D Receptor  Wnt TCF/LEF Xenobiotic AhR

23 GFP-based Reporter: Small Molecule Studies
Fluorescent Microscopy Fluorometry 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 Utility of the Cignal Reporter Assays
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 p53 Pathway Interaction Study
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
Wnt Signaling NFkB Signaling p53 Signaling ? ? E2F Signaling MAPK/JNK Signaling Hypoxia Signaling MAPK/ERK Signaling TGFβ Signaling Myc Signaling Notch Signaling

28 Constructing Signaling Networks

29 p53 Pathway Interaction Study
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 10. HIF Cignal Reporter 11. Cignal Negative Control 12. Cignal Positive Control 1 2 3 4 5 6 7 8 9 10 11 12 p53 siRNA Neg. control siRNA

31 Results: Cancer Array Application Study
Impact of p53 siRNA Treatment

32 Post-Genomics Challenge: Pathway Interactions
p53 Signaling Hypoxia Signaling MAPK/ERK Signaling Notch Signaling

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.


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