Description(s):

ATF4 Luciferase Reporter HepG2 Stable Cell Line is derived from human liver cancer, and stably expresses firefly luciferase reporter gene under the control of CARE response element. This cell line is an ideal cellular model for monitoring the activation of stress signaling pathway triggered by stimuli treatment.

Principle

ATF4 is a transcription factor that belongs to the ATF/CREB family which has the consensus binding site cAMP-responsive element (CRE). ATF4 has numerous dimerization partners. ATF4 is induced by stress signals including anoxia/hypoxia, endoplasmic reticulum stress, amino acid deprivation, and oxidative stress and subsequently, can regulate the expression of genes involved in oxidative stress, amino acid synthesis, differentiation, metastasis, and angiogenesis.

ATF4 activates transcription by binding to CARE sequences, likely as heterodimers with members of the C/EBP family. Signosis has developed ATF4 luciferase reporter stable cell line by transfecting cells with plasmids containing ATF4 luciferase reporter (contain CARE and C/EBP sequences) and hygromycin expression cassette.  The hygromycin-resistant clones were subsequently screened for Thapsigargin-induced luciferase activity.   The cell line can be used as a reporter system for monitoring the activity of ATF4 triggered by stimuli treatment.

The principle behind TF luciferase reporter.  TF luciferase reporter stable cell line utilizes artificial promoter constructs to drive luciferase expression.  The promoter region can consist of multiple repeats of a cis-element TF binding site, a DNA fragment from the promoter region of a known TF downstream gene, or a DNA fragment containing putative/known TF binding sites.  There are several ways that a TF can be activated, such as through extracellular stimuli or through intracellular signaling pathways.  Once activated, the TF translocates to the nucleus and often interacts with relevant co-factors to drive gene expression.  Once luciferase is expressed, it can generate light in an enzymatic assay and the amount of light measured is positively correlated with the level of TF activation.

 

Data

Analysis of SL-0079 activity in response to Thapsigargin treatment. The cells were seeded on a 96-well plate overnight with DMEM including 10% FBS. The cells then were treated with or without 300 nM TG (Thapsigargin) in DMEM and 10% FBS for about 18 hours. Data was read on a luminometer with a sensitivity of 3x10^-21 moles luciferase.

Literature

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