GEArray Analysis
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Cignal Reporter Assays (45 Pathways) Pathway Amino Acid Deprivation Androgen Antioxidant Response ATF6 C/EBP cAMP/PKA Cell Cycle DNA Damage EGR1 ER Stress Estrogen GATA Glucocorticoid Heat Shock Response Heavy Metal Stress Hedgehog HNF4 Hypoxia Interferon Regulation Type I Interferon Interferon Gamma KLF4 Liver X Transcription Factor ATF4/ATF3/ATF2 Androgen Receptor Nrf2 & Nrf1 ATF6 C/EBP CREB E2F/DP1 p53 EGR1 CBF/NF-Y/YY1 ER GATA GR HSF MTF1 GLI HNF4 HIF-1 IRF-1 STAT1/STAT2 STAT1/STAT1 KLF4 LXR Pathway MAPK/ERK MAPK/JNK MEF2 c-myc Nanog NFκB Notch Oct4 Pax6 PI3K/AKT PKC/Ca++ PPAR Progesterone Retinoic Acid Retinoid X Sox2 SP1 STAT3 TGFβ Vitamin D Wnt Xenobiotic Transcription Factor Elk-1/SRF AP-1 MEF2 Myc/Max Nanog NFκB RBP-Jk Oct4 Pax6 FOXO NFAT PPAR PR RAR RXR Sox2 SP1 STAT3 SMAD2/3/4 Vitamin D Receptor TCF/LEF AhR http://www.sabiosciences.com/cellassay.php -1- Sample & Assay Technologies Cignal™ Reporter Assays Cell-Based Assays for Rapidly Analyzing Pathway Signaling Activity Samuel J. Rulli, Jr., Ph.D. qPCR and Pathway Signaling Application Scientist -2- Sample & Assay Technologies 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 . -3- Sample & Assay Technologies Post-Genomics Research Challenge: Moving from a Signaling Flowchart Perspective to a Signaling Network Perspective. DNA damage Basic Signal Transduction Flowchart ATM/ATR p53 Cell cycle arrest/Apoptosis -4- Sample & Assay Technologies Post-Genomics Research Challenge: Ionizing Ionizing Radiation Radiation Chemotherapy Chemotherapy Hypoxia Hypoxia UV UV p53 Signaling F a s p53 p53 Degradation Degradation Signal Transduction Cascade A B C D JNK HIPK2 p38 CSNK1 Proteasome Proteasome Hypoxia Hypoxia Induced Induced HIF1 HIF1 Expression Expression DNA DNA Damage Damage HIF1α PTEN D R 5 P p53 Apoptosis Apoptosis ATR P ATM P p53 Chk1 P GSK3β Akt Chk2 Ub PIAS1 DNA-PK Ub Caspase PML p53 Ac BRCA1 MDM2 Cell Cell Survival Survival CAK Ac p53 P p53 P PTEN Bax PCAF Ub (~40 proteins) c-Abl BCL2 Ac Gene Gene Expression Expression p53 HDAC DNA Repair TSP1 p300 Sirt p21CIP MDM2 BAI1 GADD45 c-Fos P CyclinD1 Rb CDK2 E2F Rb 14-3-3 -5- CyclinE E2F Cell Cycle Progression Angiogenesis Angiogenesis Inhibition Inhibition CDK4 Sample & Assay Technologies 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. -6- Sample & Assay Technologies 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 -7- Sample & Assay Technologies 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 . -8- Sample & Assay Technologies Cignal Pathway Reporter System Products Primary cells, stem cells, and difficult to transfect cell lines Easy to transfect cell lines Endpoint Assays Cignal Dual-Luciferase Reporter Assays Single Pathway Assays 10-Pathway Arrays Dynamic Live Cell Assays Dynamic Live Cell Assays Endpoint Assays Cignal GFP Reporter Assays Cignal Lenti Luciferase Reporters Single Pathway Assays Single Pathway Assays -9- 10-Pathway Arrays Cignal Lenti GFP Reporters Single Pathway Assays Sample & Assay Technologies Cignal Reporter Assays (45 Pathways) Pathway Amino Acid Deprivation Androgen Antioxidant Response ATF6 C/EBP cAMP/PKA Cell Cycle DNA Damage EGR1 ER Stress Estrogen GATA Glucocorticoid Heat Shock Response Heavy Metal Stress Hedgehog HNF4 Hypoxia Interferon Regulation Type I Interferon Interferon Gamma KLF4 Liver X Transcription Factor ATF4/ATF3/ATF2 Androgen Receptor Nrf2 & Nrf1 ATF6 C/EBP CREB E2F/DP1 p53 EGR1 CBF/NF-Y/YY1 ER GATA GR HSF MTF1 GLI HNF4 HIF-1 IRF-1 STAT1/STAT2 STAT1/STAT1 KLF4 LXR - 10 - Pathway MAPK/ERK MAPK/JNK MEF2 c-myc Nanog NFκB Notch Oct4 Pax6 PI3K/AKT PKC/Ca++ PPAR Progesterone Retinoic Acid Retinoid X Sox2 SP1 STAT3 TGFβ Vitamin D Wnt Xenobiotic Transcription Factor Elk-1/SRF AP-1 MEF2 Myc/Max Nanog NFκB RBP-Jk Oct4 Pax6 FOXO NFAT PPAR PR RAR RXR Sox2 SP1 STAT3 SMAD2/3/4 Vitamin D Receptor TCF/LEF AhR Sample & Assay Technologies Cignal Lenti Assays (35 Pathways) Pathway Amino Acid Deprivation Androgen Antioxidant Response ATF6 C/EBP cAMP/PKA Cell Cycle DNA Damage EGR1 ER Stress Estrogen GATA Glucocorticoid Heat Shock Response Heavy Metal Stress Hedgehog HNF4 Hypoxia Interferon Regulation Type I Interferon Interferon Gamma KLF4 Liver X Transcription Factor ATF4/ATF3/ATF2 Androgen Receptor Nrf2 & Nrf1 ATF6 C/EBP CREB E2F/DP1 p53 EGR1 CBF/NF-Y/YY1 ER GATA GR HSF MTF1 Gli HNF4 HIF-1 IRF-1 STAT1/STAT2 STAT1/STAT1 KLF4 LXR - 11 - Pathway MAPK/ERK MAPK/JNK MEF2 c-myc Nanog NFκB Notch Oct4 Pax6 PI3K/AKT PKC/Ca++ PPAR Progesterone Retinoic Acid Retinoid X Sox2 SP1 STAT3 TGFβ VDRE Wnt Xenobiotic Transcription Factor Elk-1/SRF AP-1 MEF2 Myc/Max Nanog NFκB RBP-Jk Oct4 Pax6 FOXO NFAT PPAR PR RAR RXR Sox2 SP1 STAT3 SMAD2/3/4 Vitamin D Receptor TCF/LEF AhR Sample & Assay Technologies 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 TRE TATA box Firefly luciferase Normalization Construct CMV TATA box Renilla luciferase Constitutive transcriptional regulatory element (1) - 12 - Sample & Assay Technologies 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 - 13 - Sample & Assay Technologies 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 TRE TATA box Firefly luciferase Normalization Construct CMV TATA box Renilla luciferase Constitutive transcriptional regulatory element (1) - 14 - Sample & Assay Technologies 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 Dual-Luciferase Assay design corrects for these unwanted sources of variability. • Changes due to the designed treatments of cells • RNA interference • Overexpression • Recombinant protein/peptide/growth factor • Small molecule - 15 - Sample & Assay Technologies Dual Luciferase Format Minimizes Variability Dual-Luciferase Assays: NFkB Reporter Assay Single-Luciferase Assays: NFkB Reporter Assay 30000000 2.5 CV=0.07 Luminescence (Relative Light Units) Relative Luciferase Units CV=0.70 25000000 20000000 15000000 CV=0.69 10000000 5000000 0 2 1.5 1 CV=0.09 0.5 0 NFkB Reporter + NFkB siRNA NFkB Reporter + Neg. control siRNA NFkB Reporter + NFkB siRNA - 16 - NFkB Reporter + Neg. control siRNA Sample & Assay Technologies 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 TRE TATA box Firefly luciferase Normalization Construct CMV TATA box Renilla luciferase - 17 - Sample & Assay Technologies 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) 250 Relative Luciferase Units 0.35 Fold Induction 200 150 WHY? 100 50 0.3 0.25 0.2 0.15 0.1 0.05 0 0 Negative Control NFkB Reporter: NFkB Reporter: Stable Luc Destabilized Luc - 18 - Negative Control NFkB Reporter: NFkB Reporter: Stable Luc Destabilized Luc Sample & Assay Technologies 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 TRE TATA box Firefly luciferase Normalization Construct CMV TATA box Renilla luciferase - 19 - Sample & Assay Technologies Cignal TRE Maximize Luciferase Signal Dual-Luciferase Assays Utilizing Cignal p53 TRE 3.5 160 3 140 Relative Luciferase Units Relative Luciferase Units Dual-Luciferase Assays Utilizing Common p53 TRE 2.5 2 1.5 2.5-Fold 1 0.5 0 120 100 80 60 125-Fold 40 20 0 p53 Reporter + DMSO p53 Reporter + 1µM Doxorubicin p53 Reporter + DMSO - 20 - p53 Reporter + 1µM Doxorubicin Sample & Assay Technologies Cignal Reporter Assays: How It Works - 21 - Sample & Assay Technologies Cignal Reporter Assays (GFP) Pathway Amino Acid Deprivation Androgen Antioxidant Response ATF6 C/EBP cAMP/PKA Cell Cycle DNA Damage EGR1 ER Stress Estrogen GATA Glucocorticoid Heat Shock Response Heavy Metal Stress Hedgehog HNF4 Hypoxia Interferon Regulation Type I Interferon Interferon Gamma KLF4 Liver X Transcription Factor ATF4/ATF3/ATF2 Androgen Receptor Nrf2 & Nrf1 ATF6 C/EBP CREB E2F/DP1 p53 EGR1 CBF/NF-Y/YY1 ER GATA GR HSF MTF1 Gli HNF4 HIF-1 IRF-1 STAT1/STAT2 STAT1/STAT1 KLF4 LXR - 22 - Pathway MAPK/ERK MAPK/JNK MEF2 c-myc Nanog NFκB Notch Oct4 Pax6 PI3K/AKT PKC/Ca++ PPAR Progesterone Retinoic Acid Retinoid X Sox2 SP1 STAT3 TGFβ VDRE Wnt Xenobiotic Transcription Factor Elk-1/SRF AP-1 MEF2 Myc/Max Nanog NFκB RBP-Jk Oct4 Pax6 FOXO NFAT PPAR PR RAR RXR Sox2 SP1 STAT3 SMAD2/3/4 Vitamin D Receptor TCF/LEF AhR Sample & Assay Technologies 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. - 23 - Sample & Assay Technologies 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) - 24 - Sample & Assay Technologies 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 . - 25 - Sample & Assay Technologies 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? - 26 - Sample & Assay Technologies Post-Genomic Challenges: Pathway Interactions Wnt Signaling NFkB Signaling p53 Signaling ? ? E2F Signaling ? ? Hypoxia Signaling MAPK/JNK Signaling ? ? ? TGFβ Signaling MAPK/ERK Signaling ? ? Myc Signaling Notch Signaling - 27 - Sample & Assay Technologies Constructing Signaling Networks - 28 - Sample & Assay Technologies 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. - 29 - Sample & Assay Technologies Cignal Finder Cancer Array : p53 RNAi Study 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 1 2 4 3 5 6 7 8 9 10 11 12 p53 siRNA Neg. control siRNA 10. HIF Cignal Reporter 11. Cignal Negative Control 12. Cignal Positive Control - 30 - Sample & Assay Technologies Results: Cancer Array Application Study Impact of p53 siRNA Treatment - 31 - Sample & Assay Technologies Post-Genomics Challenge: Pathway Interactions p53 Signaling Hypoxia Signaling MAPK/ERK Signaling Notch Signaling - 32 - Sample & Assay Technologies 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 - 33 - Sample & Assay Technologies
