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Cignal Reporter Assays (45 Pathways)

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 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

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Cignal Reporter Assays (45 Pathways)

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  1. Cignal Reporter Assays (45 Pathways) PathwayTranscription Factor MAPK/ERKElk-1/SRF MAPK/JNKAP-1 MEF2MEF2 c-mycMyc/Max NanogNanog NFκB NFκB  Notch RBP-Jk Oct4Oct4  Pax6Pax6 PI3K/AKTFOXO PKC/Ca++ NFAT PPARPPAR  ProgesteronePR Retinoic Acid RAR Retinoid XRXR Sox2Sox2 SP1SP1  STAT3STAT3 TGFβSMAD2/3/4 Vitamin DVitamin D Receptor  Wnt TCF/LEF Xenobiotic AhR PathwayTranscription Factor Amino Acid DeprivationATF4/ATF3/ATF2  AndrogenAndrogen Receptor Antioxidant ResponseNrf2 & Nrf1 ATF6ATF6 C/EBPC/EBP cAMP/PKACREBCell CycleE2F/DP1 DNA Damagep53 EGR1EGR1 ER StressCBF/NF-Y/YY1 EstrogenER GATAGATA GlucocorticoidGR Heat Shock ResponseHSF Heavy Metal StressMTF1 HedgehogGLI HNF4HNF4 HypoxiaHIF-1 Interferon RegulationIRF-1 Type I InterferonSTAT1/STAT2 Interferon GammaSTAT1/STAT1 KLF4KLF4 Liver XLXR 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. 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. 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. Easy to transfect cell lines Primary cells, stem cells, and difficult to transfect cell lines Dynamic Live Cell Assays Dynamic Live Cell Assays Endpoint Assays Endpoint Assays Cignal Dual-Luciferase Reporter Assays Cignal GFP Reporter Assays Cignal Lenti Luciferase Reporters Cignal Lenti GFP Reporters Single Pathway Assays 10-Pathway Arrays Single Pathway Assays Single Pathway Assays Single Pathway Assays 10-Pathway Arrays Cignal Pathway Reporter System Products

  10. Cignal Reporter Assays (45 Pathways) PathwayTranscription Factor MAPK/ERKElk-1/SRF MAPK/JNKAP-1 MEF2MEF2 c-mycMyc/Max NanogNanog NFκB NFκB  Notch RBP-Jk Oct4Oct4  Pax6Pax6 PI3K/AKTFOXO PKC/Ca++ NFAT PPARPPAR  ProgesteronePR Retinoic Acid RAR Retinoid XRXR Sox2Sox2 SP1SP1  STAT3STAT3 TGFβSMAD2/3/4 Vitamin DVitamin D Receptor  Wnt TCF/LEF Xenobiotic AhR PathwayTranscription Factor Amino Acid DeprivationATF4/ATF3/ATF2  AndrogenAndrogen Receptor Antioxidant ResponseNrf2 & Nrf1 ATF6ATF6 C/EBPC/EBP cAMP/PKACREBCell CycleE2F/DP1 DNA Damagep53 EGR1EGR1 ER StressCBF/NF-Y/YY1 EstrogenER GATAGATA GlucocorticoidGR Heat Shock ResponseHSF Heavy Metal StressMTF1 HedgehogGLI HNF4HNF4 HypoxiaHIF-1 Interferon RegulationIRF-1 Type I InterferonSTAT1/STAT2 Interferon GammaSTAT1/STAT1 KLF4KLF4 Liver XLXR

  11. Cignal Lenti Assays (35 Pathways) PathwayTranscription 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 Sox2Sox2 SP1  SP1  STAT3  STAT3  TGFβ SMAD2/3/4 VDRE  Vitamin D Receptor  Wnt TCF/LEF Xenobiotic AhR PathwayTranscription Factor Amino Acid Deprivation  ATF4/ATF3/ATF2 Androgen  Androgen Receptor  Antioxidant Response  Nrf2 & Nrf1  ATF6ATF6 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

  12. Pathway-targeted transcriptional regulatory elements (45) Reporter Construct TATA box TATA box TRE Firefly luciferase CMV Renilla luciferase Normalization Construct Constitutive transcriptional regulatory element (1) 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. Pathway-targeted transcriptional regulatory elements (45) Reporter Construct TATA box TATA box TRE Firefly luciferase CMV Renilla luciferase Normalization Construct Constitutive transcriptional regulatory element (1) 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? Dual-Luciferase Assay design corrects for these unwanted sources of variability. • 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

  16. Dual Luciferase Format Minimizes Variability NFkB Reporter + NFkB siRNA NFkB Reporter + Neg. control siRNA Dual-Luciferase Assays: NFkB Reporter Assay Single-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

  17. Reporter Construct TATA box TATA box TRE Firefly luciferase CMV Renilla luciferase Normalization Construct 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 Signal:Noise Ratio WHY? Modified Luciferase: NFkB Reporter Signal Following TNF Induction Modified Luciferase: Background NFkB Reporter Signal (no treatment post-transfection) Negative Control NFkB Reporter: Stable Luc NFkB Reporter: Destabilized Luc Negative Control NFkB Reporter: Stable Luc NFkB Reporter: Destabilized Luc

  19. Pathway-targeted transcriptional regulatory elements (45) Reporter Construct TATA box TATA box TRE Firefly luciferase CMV Renilla luciferase Normalization Construct 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 Common p53 TRE 2.5-Fold 125-Fold p53 Reporter + DMSO p53 Reporter + 1µM Doxorubicin 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) PathwayTranscription Factor MAPK/ERK Elk-1/SRF MAPK/JNK AP-1 MEF2  MEF2  c-myc Myc/Max NanogNanog 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 Sox2Sox2 SP1  SP1 STAT3  STAT3  TGFβ SMAD2/3/4 VDRE  Vitamin D Receptor Wnt TCF/LEF Xenobiotic AhR PathwayTranscription Factor Amino Acid Deprivation  ATF4/ATF3/ATF2  Androgen  Androgen Receptor  Antioxidant Response  Nrf2 & Nrf1 ATF6  ATF6  C/EBP C/EBP cAMP/PKA CREBCell 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

  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 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

  27. Post-Genomic Challenges: Pathway Interactions p53 Signaling Wnt Signaling NFkB Signaling ? ? ? MAPK/JNK Signaling E2F Signaling ? ? Hypoxia Signaling ? ? MAPK/ERK Signaling TGFβ Signaling ? ? Myc Signaling Notch Signaling

  28. Constructing Signaling Networks

  29. Application 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. p53 Pathway Interaction Study

  30. Cignal Finder Cancer Array : p53 RNAi Study p53 siRNA Neg. control siRNA • 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 2 4 1 8 9 10 11 12 5 3 6 7

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

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