| Description |
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The Toxicity Reporter Array is the first commercial reporter array that
allows you a comprehensive view of stress and toxicity response signaling
pathways. Don't rely on guesswork to predict gene or drug function. Use the
Cignal Finder Toxicity Array to pinpoint the pathways regulated by the genes
or drugs your laboratory is studying.
Learn how the Toxicity Reporter Array works
The Cignal Finder Toxicity 10-Pathway Reporter Array is ideally suited for
discovering:
- siRNA/shRNA/miRNA phenotypes
- Biological responses to chemical compounds
- Mechanisms of action of proteins, peptides, and ligands
For more information about the Cignal Finder 10-Pathway Reporter Arrays,
please visit the Cignal Finder Array
home page.
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| Pathway Listing and Performance Data |
| Tube/Column |
Pathway |
Transcription Factor |
| 1 |
p53/DNA Damage |
p53 |
| 2 |
Hypoxia |
HIF1A |
| 3 |
NFκB |
NFκB |
| 4 |
Glucocorticoid Receptor |
GR |
| 5 |
Cell Cycle/pRB-E2F |
E2F/DP1 |
| 6 |
MAPK/ERK |
ELK1/SRF |
| 7 |
MAPK/JNK |
AP1 |
| 8 |
PKC/Ca++ |
NFAT |
| 9 |
TGFβ |
SMAD2/SMAD3/SMAD4 |
| 10 |
Myc/Max |
Myc/Max |
| 11 |
Cignal negative control |
|
| 12 |
Cignal positive control |
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Learn more about the 10 pathways
View each reporter's transcriptional
response element sequence
Eukaryotic cells are constantly subjected to environmental and
intracellular stress. Sources of cellular stress and toxicity include
physical and biochemical agents, as well as cellular processes, which lead
to oxidative stress, DNA damage, osmotic shock, nutrient depletion, heat
shock, and oncogene overexpression. In order to survive and maintain genomic
integrity in the face of these toxic onslaughts, cells utilize numerous
protective mechanisms. The Cignal Stress and Toxicity Reporter Array
includes 10 Cignal Reporter Assays for 10 signaling pathways that respond to
protect cells from agents of cytotoxicity and cellular stress.
The tumor suppressor p53 is the classic "guardian of the
genome", serving as a critical sensor of genotoxic stress, hypoxia,
cytokine signaling, viral infections, and oncogene overexpression. One of
the many downstream targets of the p53 transcription factor is the pRb/E2F
pathway. Several targets of the E2F transcription factors are
important regulators of both DNA synthesis and DNA repair. The
hypoxia-responsive transcription factor HIF1α activates genes leading
to p53 stabilization. HIF1α also induces the xenobiotic response, which
leads to the expression of xenobiotic metabolizing enzymes. The xenobiotic
response is also regulated by the nuclear hormone receptors the Aryl
hydrocarbon Receptor (AhR) and the Glucocorticoid Receptor (GR). In addition
to its role in the xenobiotic response, Glucocorticoid Receptors also
activate genes leading to the inhibition of the the TGFβ and MAPK signaling
pathways. The MAPK/ERK and MAPK/JNK pathways are classic stress-induced
pathways. Oxidative stress, membrane damage, osmotic shock, and heat shock
responses all involve signaling through MAPK signal transduction pathways.
One of the many transcription factors activated in response to MAPK
stress-induced signaling is c-Myc. Osmotic shock and membrane damage impact
the calcium homeostasis of the cell, leading to changes in the activities of
the cAMP/PKA and PKC/Ca++ signaling pathways. The NFκB transcription factor
family members are activated in response to several forms of stress. These
include cytokines, free radicals, uv irradiation, oxidative stress, and
bacterial and viral antigens.
Performance Data
Identify stress and toxicity responses induced by a chemical compound
The Cignal Finder Toxicity 10-Pathway Reporter Array can be used early in
the drug discovery process to determine the effects of a screened compound
on stress- and toxicity-related signaling pathways.
The Cignal Finder Toxicity 10-pathway Reporter Array showed that 300 µM
CoCl2 activates p53, hypoxia and MAPK/JNK (classical stress) signaling in
HepG2 cells.
HepG2 cells were reverse transfected with the Cignal Finder Stress and
Toxicity 10-Pathway Reporter Array. After 16 hours of transfection, medium was
changed to assay medium (Opti-MEM containing 0.5% of fetal bovine serum, 1%
NEAA, 100 U/ml Penicillin and 100 µg/ml Streptomycin). After 40 hours of
transfection, cells were treated with 300 µM CoCl2. Dual-luciferase assays were
performed after 18 hours of treatment, and results are expressed as fold
change. The fold change was calculated by dividing the normalized luciferase
activities of each pathway-focused reporter treated with CoCl2 by the
normalized luciferase activity of the same pathway-focused reporter that was
not treated. Experiments were done in quadruplicates, and the standard
deviations are indicated.
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| How It Works
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Each Reporter Array includes 10 Cignal Reporter Assays and two controls in
either tube or plate format. All reporter assays are based on dual-luciferase
technology. Each reporter consists of a mixture of a pathway-focused
transcription factor-responsive firefly luciferase construct and a
constitutively expressing Renilla luciferase construct.
Dual-luciferase results are calculated for each transfectant. The change in
the activity of each signaling pathway is determined by comparing the normalized
luciferase activities of the reporter in treated versus untreated transfectants.
The identically treated negative control serves as a specificity control. The
positive control serves as a control for transfection efficiency, by monitoring
GFP expression, as well as a positive control for both the firefly and Renilla
luciferase assays.
Simple Procedure:
- Transfect Cignal Reporter Assays and test nucleic acids into cells
- Treat with protein, peptide, or small molecule of interest
- Perform reporter quantitation using luciferase activity assays
Cignal Finder Array - Tube Format
The tube format of the Cignal Finder Arrays are delivered in 12-tube
strips, along with important negative and positive controls. The assays
are used right out of the box for the transfection or reverse
transfection of the reporter assays into your cell lines of interest.
Cignal Finder Array - Plate Format
The plate format of the Cignal Finder Arrays are delivered in a 96-well
cell culture plate. Each reporter and control assay is dried down in
each column of the plate (8 wells per assay).
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| Useful Links |
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| Related Products |
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| Brief Protocol: For Experienced
Users |
First time users, please refer to the complete
protocol in the Cignal Reporter Assays User Manual.
- Dilute transfection-ready reporter, negative control, and positive
control construct formulations.
- Dilute relevant test nucleic acids (siRNA, shRNA, miRNA, expression
vector).
- Prepare appropriate combinations of reporter constructs, controls,
and test nucleic acids.
- Transfect plasmid mixtures separately into replicate wells of your
cell line of interest using an optimized transfection procedure for
the cell line under study.
- If applicable, 16 to 24 hours post-transfection, treat the
transfected cells with test proteins, peptides, or compounds of
interest.
- Two (2) to three (3) days post-transfection, assay the activities of
the signaling pathways under study, utilizing the dual luciferase
assay.
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