Cignal Finder Reporter Arrays

Cignal Finder Reporter Arrays can be used in many applications.

Identification of optimal drug doses

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. Cell-based luciferase reporter assays were used to measure 10 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 10-Pathway Reporter Array was used to determine the lowest effective concentration of Tunicamycin without activating cellular stress pathways. Results showed that 0.125 µg/ml Tunicamycin is the lowest effective non-toxic dose for glycoprotein synthesis inhibition (see figure "Identification of optimal drug dose").

Cell response to 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 affected by p53 knockdown. The Cancer 10-Pathway Reporter Array can help to identify key cancer signaling pathways modulated by p53 knockdown (see figure "Signaling pathways affected by p53 knockdown").

Results showed that knockdown of p53 gene expression downregulates p53 signaling, while upregulating Notch, hypoxia, and MAPK/ERK signaling in HEK-293H cells. 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.

Measurement of signaling in response to cytokines

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

The Immune Response 10-Pathway Cignal Finder Reporter Array reveals that TNFα activates the NFkB and MAPK/JNK signaling pathways in HeLa cells (see figure Signaling pathways affected by TNFalpha).

Each array includes 10 Cignal Reporter Assays and 2 controls in either a transfection-ready 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.

The procedure is comprised of 3 simple steps:

• Transfect Cignal Finder Reporter Assays and test nucleic acids into cells 
• Treat with siRNA, protein, peptide, or small molecule of interest 
• Perform reporter quantitation using luciferase activity assays

Cignal Finder Reporter Array: Tube Format

Cignal Finder Reporter Arrays in tube format are delivered in 12-tube strips, along with positive and negative controls. The assays are immediately ready for use in the transfection or reverse transfection of the reporter assays into the cell lines of interest (see flowchart "Cignal Finder Array, tube format procedure").

Cignal Finder Reporter Array: Plate Format

Cignal Finder Reporter Arrays in plate format 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; see flowchart "Cignal Finder Array, plate format procedure").

Cignal Reporter Arrays are highly suited for research into phenotypes associated with RNAi or overexpression experiments, biological responses to small molecules or compounds, and mechanisms of action of proteins, peptides, and ligands.