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Cignal Reporter Array Applications

Find the signaling pathways that respond to gene overexpression/knockdown or chemical treatment. Cignal Finder Reporter Arrays are dual-luciferase assays that enable comprehensive analysis of multiple pathways in a single experiment.

Pharmacology Example

Identify Optimal Doses of a Drug Treatment

Toxicity is one of the primary reasons potential drug candidates fail during development. Identifying potential toxicity and the cellular response to a given concentration or compound can benefit future studies. Toxic compounds or dosages activate various stress response pathways. SABiosciences has developed cell-based luciferase reporter assays to measure ten stress response pathways using a single 96-well plate.

Tunicamycin inhibits the enzyme GlcNAc phosphotransferase (GPT) and induces Endoplasmic Reticulum (ER) stress. In this study, the Stress and Toxicity Reporter Array was used to determine the lowest effective concentration of Tunicamycin without activating cellular stress pathways. Results from Cignal Reporter Stress and Toxicity Response 10-Pathway Reporter Array revealed that 0.125 µg/ml of Tunicamycin is the lowest effective non-toxic dose for glycoprotein synthesis inhibition.

HepG2 cells were reverse transfected with each reporter assay and the controls on the Stress & Toxicity 10-Pathway Reporter Array plate. Sixteen hours after carrying out the reverse transfection, the medium was changed to complete medium (DMEM containing 10% of fetal bovine serum, 1% NEAA, 100 U/ml Penicillin and 100 g/ml Streptomycin). After 30 hours of transfection, cells were treated with increasing dosages of tunicamycin (0 4 micrograms / milliliter). After 48 hours of transfection, the dual-luciferase assay was performed and results are expressed as fold change. The fold change was calculated by dividing the normalized luciferase activities of each treated pathway-focused reporter by the normalized luciferase activity of the untreated pathway-focused reporter. Experiments were performed in triplicate.

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For a detailed description of the protocol and other technical tips: User Manual

Cancer Biology Example

How cells respond to the loss of a Cancer Signaling Pathway

The most well studied tumor suppressor gene is p53. To understand more about the biological function of p53, it is important to measure the signaling pathways perturbed by knockdown of p53. The Cignal Finder Cancer 10-Pathway Reporter Array provides a vital tool to identify the key cancer signaling pathways modulated by knock down of p53.

The Cignal Finder Cancer 10-Pathway Reporter Array showed that the knock down of p53 gene expression down-regulates p53 signaling, while up-regulating Notch, hypoxia and MAPK/ERK signaling in HEK-293H cells. Interestingly, Notch signaling is known to be frequently deregulated in human malignancies. Activation of Notch signaling by p53 RNA interference suggests that Notch may function as a proto-oncogene.

HEK-293H cells were co-transfected with either p53 siRNA or a negative control siRNA, in combination with each reporter assay and the negative control from the Cancer 10-Pathway Reporter Array plate. Sixteen hours after carrying out the reverse transfection, medium was changed to complete medium (DMEM containing 10% of fetal bovine serum, 1% NEAA, 100 U/ml Penicillin and 100 µg/ml Streptomycin). After 48 hours of transfection, the dual-luciferase assay was performed and results are expressed as fold change. 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. Experiments were done in quadruplicates, and the standard deviations are indicated.

Return to Cignal Array Home page

For a detailed description of the protocol and other technical tips: User Manual

Immunology Example

Measure Signaling in response to Cytokines

Tumor necrosis factor-alpha (TNF-α) is a pleiotropic inflammatory cytokine. It is important to determine the key immunology signaling pathways modulated by TNF-α. The Cignal Finder Immune Response 10-Pathway Reporter Array can provide valuable information about the signaling pathways involved in the biological response to TNF-α.

The Immune Response 10-pathway Reporter Array reveals that TNF-α activates the NFκB and MAPK/JNK signaling pathways in HeLa cells.



HeLa cells were reverse transfected with the Cignal Immune Response 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 32 hours of transfection, cells were treated with 5 ng/ml of TNF-α, or were left untreated. After 6 hours of treatment, dual-luciferase assays were performed 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 TNF-α by the normalized luciferase activity of the respective untreated pathway-focused reporter. Experiments were done in quadruplicates, and the standard deviations are indicated.

Return to Cignal Array Home page

For a detailed description of the protocol and other technical tips: User Manual