Description:

Principle:

STAT1 exerts a complex array of functions on both tumor cells and the immune system and is usually considered as a tumor suppressor. STAT1 is a central mediator of type II (gamma) IFNs, a family of multifunctional secreted proteins involved in cell growth regulation and antiviral and immune defense. IFN-gamma (IFNγ), through JAK1 and JAK2, mainly triggers prolonged STAT1 activation that induces gene expression by binding to gamma-activated sequences (GAS). This cell line can be used to monitor the activation of Stat1 in response to the different stimuli, such as IFNgamma.

The cell line was established by transfectionusing apTA-Stat1-luciferase reporter vector, which contains 4 repeats of Stat1 binding sites, a minimal promoter upstream of the firefly luciferase coding region,along with hygromycin expression vector followed by hygromycin selection.The hygromycin resistant clones were subsequently screened for oncostatin induced luciferase activity. The clone with the highest fold induction (50 fold) was selected and expanded to produce this stable cell line.

Principle behind TF luciferase reporter. TF luciferase reporter stable cell line utilizes artificial promoter constructs to drive luciferase expression. The promoter region can consists 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 HeLa/STAT1-luciferase activity.The cells were seeded on a 96-well plate for overnight and then cells were treated with or without 30ng/ml interferon-gamma in DMEM and 0.1% FBS for 16 hours. Interferon-gamma induces more than 25 fold increase in luciferase activity when compared to untreated cells.