CIGNAL REPORTER ASSAYS & ARRAYS

Transcription

1 CIGNAL REPORTER ASSAYS & ARRAYS TM Cell-Based for Measuring Activity Cancer Cell Cycle Cytokines / Inflammation Neuroscience Stem Cell / Development Toxicology Focus on your

2 CIGNAL TM REPORTER ASSAY SYSTEM CIGNAL REPORTER ASSAYS The Cignal are cell-based transcription factor reporters for monitoring cellular signaling. The Cignal provide a rapid, sensitive, and quantitative measurement of signal pathway activation by measuring the activities of downstream transcription factors. Cignal assays are offered as either dual-luciferase or green fluorescent protein (GFP) reporter genes. Every reporter assay is individually engineered to exhibit outstanding sensitivity, specificity, and signal-to-noise ratio. The Cignal s are available in two delivery formats, transfection-ready DNA-based constructs or high efficiency transduction-ready lentiviral particles. Taken together, these reporter assays are valuable tools for understanding gene function, as well as determining the mechanism of action for proteins, peptides, or small molecule compounds. REPORTER GENES LUCIFERASE REPORTERS Exceptional Sensitivity and Specificity High Reproducibility High Signal-to-Noise Ratio GFP REPORTERS Cell Resolution Dynamic Live Cell Assay Flexible Readout (FACS, Microscopy, or Fluorometer) Cignal Selection Guide DELIVERY TECHNOLOGIES DNA-BASED CONSTRUCTS Ready-to-Transfect Optimized Formulations for Transfection Efficiency LENTIVIRAL PARTICLES Study Activation in Cells Refractory to Transfection Generate Sensor Cell Lines Cignal Protocol Overview PATHWAY REPORTER ASSAYS Luciferase OR GFP Easy-to-Transfect Cell Lines Primary Cells, Stem Cells, and Difficult-to-Transfect Cell Lines Firefly Luciferase Tandem TRE GFP Renilla Luciferase CMV Tandem TRE Endpoint Live Cell Endpoint Live Cell TRANSFECTION / TRANSDUCTION DNA-based Dual-Luciferase DNA-based GFP Lenti Luciferase s Lenti GFP s TREATMENT ENT ASSAY READOUT 1- Arrays 1- Arrays Measure Signaling Activity with Dual-Luciferase Assay Flow Cytometry High Content Screening Fluorescent Microscopy Fluorometer Support@SABiosciences.com

3 CIGNAL: Performance High Sensitivity Delivery Power Relative Luciferase Units Negative Control Cignal s Monitor Activation Utilizing Luciferase Negative Control + 5 ng/ml +.5 ng/ml +.5 ng/ml + 5 ng/ml + 5 ng/ml Cignal Showed That Tumor Necrosis Factor Alpha (TNFα) Activated Signaling Activity in a Dose-dependent Manner. HEK-293 cells were transiently transfected with the reporter, negative or positive control. A Renilla luciferase expression plasmid was used as a transfection efficiency control. After 24 hours of transfection, cells were treated with increasing doses of recombinant TNFα for 24 hours. Cells were lysed and assayed for luciferase activity. Relative luciferase activity is shown as the mean (± S.D.) of three independent experiments. Relative Luciferase Units Lenti s Measure Activity in Primary Cells Cignal Lenti NFAT (luciferase) + DMSO Cignal Lenti (luciferase) + 1 ng/ml PMA +.5 μm Ionomycin Cignal Lenti NFAT Measured PKA/Ca ++ Activity in Human Primary Cells [Normal Human Pulmonary Artery Smooth Muscle Cells, (PASMC)]. Cignal Lenti NFAT reporter [4 1 5 TU] and Cignal Lenti Renilla control [1 1 5 TU] co-transduced approximately 1, PASMC cells (24 hours before transduction 5, cells were plated per well of 96-well plate). The Renilla luciferase control was used as a transduction efficiency control. After 48 hours of transduction, medium was changed to assay medium. After 54 hours of transduction, cells were treated with 1 ng/ml PMA and.5 μm ionomycin for 18 hours. Cells were lysed and assayed for luciferase activity. Relative luciferase activity is shown as the mean (±S.D.) of three independent experiments. Visualize Activity Stable Cell Line Generation Cignal GFP s Provide A Fluorescent Readout of Activity Lenti s Establish Sensor Cell Lines A C B D Relative Fluorescence Units E Relative Luciferase Units P1 w/o 1 ng/ml P5 w/o 1 ng/ml P1 w/o 1 ng/ml P15 w/o 1 ng/ml SRE-GFP SRE-GFP + 1% Serum + 1 ng/ml PMA Cignal SRE-GFP Measured Activation of the ERK. HEK-293 cells were transfected with the Cignal SRE-GFP reporter or the negative control. After 24 hours, cells were treated with 1 ng/ml PMA and 1% serum. After 18 hours of treatment, bright field and fluorescent images were taken of the cultures transfected with the negative control (A and B, respectively) and the Cignal SRE-GFP reporter (C and D, respectively). After imaging the cells, the fluorescence was measured using a fluorometer. The relative fluorescence is shown as the mean (± S.D.) of three independent experiments (E). Generation of Sensor Cell Line Using Cignal Lenti s. Cignal Lenti reporter was used to develop a stable sensor cell line for the study of signal transduction. The sensor cell line was developed by transduction of HEK-293 cells with a Cignal Lenti reporter, followed by selection of a clonal population that maintained stable chromosomal integration of the lentiviral vector provirus and responded strongly to stimuli known to activate the pathway. The generation of a stable HEK-293 sensor cell line was confirmed by testing the responsiveness of the cell line toward 1 ng/ml of TNFα protein after the 1st (P1), 5th (P5), 1th (P1), and 15th (P15) passage of the cell line. Stimulation of the pathway by TNFα results in a 1-fold increase in expression of the reporter gene even after two months of culture.

4 CIGNAL FINDER 1-PATHWAY REPORTER ARRAYS Measure the Activities of 1 Signaling s in One Experiment Cignal Finder Arrays. The first commercial product that enables a comprehensive analysis of multiple pathways (up-to ten) in a single experiment. The Cignal Finder Arrays simultaneously measure the activities of 1 cell signaling pathways known to play critical roles in various biological processes. Each reporter array includes 1 Cignal and two controls in either a tube or plate format. All reporter assays utilize the sensitive dual luciferase reporters, which consist of a mixture of a pathway-focused transcription factor-responsive firefly luciferase construct and a constitutively expressing Renilla luciferase construct. SABiosciences has developed four focused arrays (Cancer, Immune Response, Development, and Toxicity) that facilitate pathway identification in response to sirnas, proteins, or small molecules. ANATOMY OF A CIGNAL FINDER 1-PATHWAY ARRAY Cignal Cancer Array in Tube Format Cignal Cancer Array in Plate Format Column 1. p53 Cignal Column 2. RBP-Jκ Cignal Column 3. TCF/LEF Cignal Column 4. E2F Cignal Column 5. AP-1 Cignal Column 6. Elk-1/SRF Cignal Column 7. SMAD Cignal Column 8. Cignal Column 9. Myc Cignal Column 1. HIF Cignal Column 11. Cignal Negative Control Column 12. Cignal Positive Control NOTE: Each color denotes an individual pathway-specific luciferase reporter. Comprehensive Analysis Cignal Arrays Measure Activation and Crosstalk Activation Fold Change TCF/LEF RBP-Jκ p53 SMAD E2F Myc HIF Elk-1/SRF AP-1 Cignal Inhibition of the p53 Signaling by p53 sirna Treatment Results in Activation of the Notch, Hypoxia, and MAPK/ERK s. HEK-293 cells were co-transfected with either p53 sirna or a negative control sirna, in combination with each reporter assay or the negative control from the Cancer 1- Array plate. Sixteen hours after carrying out the reverse transfection, medium was changed to complete medium. After 48 hours of transfection, the cells were lysed and assayed for luciferase activity. The fold change was calculated by dividing the normalized luciferase activities of each pathway-focused reporter co-transfected with p53 sirna by the normalized luciferase activity of each pathway-focused reporter co-transfected with the negative control sirna. Fold change in pathway activation is shown as the mean (± S.D.) of four independent experiments. USA TEL: FAX: INTERNATIONAL TEL: FAX:

5

6 POPULAR CIGNAL PATHWAY REPORTERS CANCER CELL CYCLE CYTOKINES / INFLAMMATION NEUROSCIENCE STEM CELL / DEVELOPMENT TOXICOLOGY p53 PI3K/AKT Notch Oct4 Heat Shock Retinoic Acid EGR1 C/EBP Estrogen Receptor Myc ERK SP1 E2F IFNγ IFNα/β TGFβ PPAR camp/pka CREB MEF2 PKC/Ca ++ Wnt STAT3 GATA Hedgehog Antioxidant Response Hypoxia JNK ER Stress (Formerly SuperArray Bioscience Corporation) 6951 Executive Way, Frederick, MD 2173 USA Phone: Fax: support@sabiosciences.com 29 SABiosciences Corporation All Rights Reserved. Cignal, Cignal Finder, SureFECT, SureENTRY, Focus on Your, SABiosciences and the SA logo are trademarks of SABiosciences Corporation. Dual-Luciferase is a registered trademark of Promega Corporation.