Kit Contents:

Component	Specification	Concentration (total volume)
HIF Reporter	A mixture of inducible HIF-responsive firefly luciferase construct and constitutively expressing Renilla luciferase construct (40:1).	(100 ng/µl; 500 µl)*
Negative control	A mixture of non-inducible firefly luciferase construct and constitutively expressing Renilla luciferase construct (40:1).	(100 ng/µl; 500 µl)
Positive control	A mixture of constitutively expressing GFP, constitutively expressing firefly luciferase, and constitutively expressing Renilla luciferase constructs (40:1:1).	(100 ng/µl; 250 µl)

* Supplied material provides sufficient reporter for 500 assays, using recommended 96-well plate transfection protocol. The number of assays per kit is a function of the assay plate format used (refer to Cignal Reporter Assay User Manual).

Storage Conditions: The Cignal reporter assay constructs are shipped ambient. Store all tubes at -20  ºC.

1. Dilute transfection-ready reporter, negative control, and positive control construct formulations.
2. Dilute relevant test nucleic acids (siRNA, shRNA, miRNA, expression vector).
3. Prepare appropriate combinations of reporter constructs, controls, and test nucleic acids.
4. Transfect plasmid mixtures separately into replicate wells of your cell line of interest using an optimized transfection procedure for the cell line under study.
5. If applicable, 16 to 24 hours post-transfection, treat the transfected cells with test proteins, peptides, or compounds of interest.
6. Two (2) to three (3) days post-transfection, assay the activities of the signaling pathways under study, utilizing the dual luciferase assay.

The Cignal Reporter Assays include pre-formulated, transfection-ready reporter, negative control, and positive control. The transcription factor reporter and negative control are transfected and subjected to experimental treatments, in parallel. Dual-luciferase results are calculated for each transfectant. The impact of the experimental treatments is determined by comparing the normalized luciferase activities of the reporter to the identically treated negative control, across the complete treatment regimen. 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.

General performance

Average maximum response rate = 4
Average Z' factor at maximum response rate = 0.80
Average coefficient of variation (CV%) = 12.4%

Excellent signal to noise ratio

Cignal HIF reporter assay showed activation of hypoxia pathway: HepG2 cells were transfected with HIF reporter, negative control and positive control (for transfection protocol refer our user manual). After 16 hours of transfection, medium was changed to assay medium (Opti-MEM + 0.5% FBS + 0.1mM NEAA + 1mM Sodium pyruvate + 100 U/ml penicillin + 100 µg/ml streptomycin). After 28 hours of transfection, cells were treated with 100µM and 250µM of CoCl2 for 18 hours. Dual Luciferase assay was performed, and promoter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were done in triplicates, and the standard deviation is indicated. Cignal HIF reporter measured 4 fold increase in HIF1 transcription activity and, in turn, showed activation of hypoxia pathway by CoCl2.

The cellular response to O2 (oxygen) is a central process in animal cells and figures prominently in the pathophysiology of several diseases, including cancer, cardiovascular disease, and stroke. This process is coordinated by the HIF (Hypoxia-Inducible Factor) and its regulator, the pVHL (Von Hippel-Lindau tumor suppressor protein). HIF1 is a basic helix-loop-helix transcription factor that transactivates genes encoding proteins that participate in homeostatic responses to hypoxia. It induces expression of proteins controlling glucose metabolism, cell proliferation, and vascularization. Several genes involved in cellular differentiation are directly or indirectly regulated by hypoxia. These include Epo (Erythropoietin), LDHA (Lactate Dehydrogenase-A), ET1 (Endothelin-1), transferrin, transferrin receptor, VEGF (Vascular Endothelial Growth Factor), Flk1, FLT1 (Fms-Related Tyrosine Kinase-1), PDGF-Beta (Platelet-Derived Growth Factor-Beta), bFGF (basic Fibroblast Growth Factor), and others genes affecting glycolysis (Ref.1).

HIF1 consists of a heterodimer of two basic helix-loop-helix PAS (Per-ARNT-Sim) proteins, HIF1-Alpha, and HIF1-Beta. HIF1-Alpha accumulates under hypoxic conditions whereas HIF1-Beta is constitutively expressed. HIF1-Alpha is an important mediator of the hypoxic response of tumor cells and controls the up-regulation of a number of factors important for solid tumor expansion including the angiogenic factor VEGF. HIF1-Beta is the ARNT (Aryl hydrocarbon Receptor Nuclear Translocator), an essential component of the xenobiotic response (Ref.2).

In the presence of O2, HIF is targeted for destruction by an E3 ubiquitin ligase containing the pVHL. Human pVHL binds to a short HIF-derived peptide when a conserved proline residue at the core of this peptide is hydroxylated. The human genome contains EGL9 (Egg Laying Nine-9) homologues that are named EGLN1, EGLN2, and EGLN3 (also called PHD2, PHD1, and PHD3 (Prolyl Hydroxylase Domain–Containing Proteins) respectively). Prolyl hydroxylase post-translationally modifies HIF1-Alpha, allowing it to interact with the VHL complex. Prolyl hydroxylase contains an iron moiety, so iron chelation inhibits this activity. All three proteins of Prolyl hydroxylase can hydroxylate HIF1-Alpha at one of two proline sites within the ODD (Pro-402 and Pro-564). Analogous prolyl residues are present in HIF2-Alpha and HIF3-Alpha. In the presence of oxygen, the EGLN proteins are active and hydroxylate the ODD domain of HIF1-Alpha, which allows pVHL to bind and polyubiquitinate HIF (Ref.3). VHL is part of a larger complex that includes Elongin-B, Elongin-C, Cul2, RBX1 (Ring-Box 1) and a ubiquitin-conjugating enzyme (E2). This complex, together with a ubiquitin-activating enzyme (E1), mediates the Ub (Ubiquitylation) of HIF1-Alpha. The Ub modification targets HIF1-Alpha for degradation, which can be blocked by proteasome inhibitors. Under hypoxic conditions the HIF1-Alpha subunits are not recognized by pVHL, and they consequently accumulate and dimerize with HIF1-Beta and translocates to the nucleus, where they interacts with cofactors such as CBP (CREB Binding Protein)/p300 and the Pol II (DNA polymerase II) complex to bind to HREs (Hypoxia-Responsive Element) and activate transcription of target genes. HIF1-Alpha-activated genes include VEGF, which promotes angiogenesis; GLUT1 (Glucose Transporter-1), which activates glucose transport; LDHA (Lactate Dehydrogenase), which is involved in the glycolytic pathway; and Epo, which induces erythropoiesis. HIF1-Alpha also activates transcription of NOS (Nitric Oxide Synthase), which promotes angiogenesis and vasodilation. ARNT2 and MOP3 (Member of Pas superfamily-3) are other proteins that have been shown to heterodimerize with HIF1-Alpha (Ref.4). HIF1-Alpha can also be regulated by ERK2, which phosphorylate HIF1-Alpha. HIF1-Alpha also associates with the molecular chaperone HSP90 (Heat Shock Protein-90). HSP90 antagonists also inhibited HIF1-Alpha transcriptional activity and dramatically reduced both hypoxia-induced accumulation of VEGF mRNA and hypoxia-dependent angiogenic activity. Recently, a factor inhibiting HIF1-Alpha activation, FIH (Factor Inhibiting HIF1-Alpha), has been described, representing a further level of HIF regulation.

Hypoxia also induces p53 protein accumulation. p53 directly interacts with HIF1-Alpha and limits hypoxia-induced expression of HIF1-Alpha by promoting MDM2-mediated ubiquitination and proteasomal degradation under hypoxic conditions. Furthermore, the degradation of HIF1-Alpha by p53 in a hypoxic condition is inhibited by direct interaction with the JAB1 (Jun Activation domain Binding protein-1) and the ODD domain by blocking the interaction with p53. HIF1-Alpha also associates with HNF4alpha2 (Hepatocyte Nuclear Factor-4-Alpha 2), which activates the Epo gene in concert with HIF1-Alpha in response to hypoxic conditions. Hypoxia contributes significantly to the pathophysiology of major categories of human disease, including myocardial and cerebral ischemia, cancer, pulmonary hypertension, congenital heart disease and chronic obstructive pulmonary diseases (Ref.5).

References:

1. Jiang BH, Zheng JZ, Leung SW, Roe R, Semenza GL.
Transactivation and inhibitory domains of hypoxia-inducible factor 1alpha. Modulation of transcriptional activity by oxygen tension.
J. Biol. Chem. 1997 Aug 1;272(31):19253-60.
PubMed ID: 9235919           

2. John F. O'Rourke, Ya-Min Tian, Peter J. Ratcliffe, and Christopher W. Pugh
Oxygen-regulated and transactivating domains in endothelial PAS protein 1: comparison with hypoxia-inducible factor-1alpha.
J. Biol. Chem. 1999 Jan 22;274(4):2060-71.
PubMed ID: 9890965

3. Masson N, Willam C, Maxwell PH, Pugh CW, Ratcliffe PJ
Independent function of two destruction domains in hypoxia-inducible factor-alpha chains activated by prolyl hydroxylation.
EMBO J. 2001 Sep 17;20(18):5197-206.
PubMed ID: 11566883

4. Damert A, Ikeda E, Risau W.
Activator-protein-1 binding potentiates the hypoxia-induciblefactor-1-mediated hypoxia-induced transcriptional activation of vascular-endothelial growth factor expression in C6 glioma cells.
Biochem. J. 1997 Oct 15;327 ( Pt 2):419-23.
PubMed ID: 9359410

5. Semenza GL.
Hypoxia-inducible factor 1: oxygen homeostasis and disease pathophysiology.
Trends Mol. Med. 2001 Aug;7(8):345-50
PubMed ID: 11516994

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