Optimization of Experimental Variables Influencing Apoptosome Biosensor in HEK293T Cells

Sensors

Saman Hosseinkhani¹  Azarakhsh Oladzad¹  Maryam Nikkhah² 

[1] Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran P.O. Box 14115-175, Iran;Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran P.O. Box 14115-175, Iran;

关键词: apoptosis;    apoptosome;    luciferase complementary assay;   

DOI  :  10.3390/s20061782

来源: DOAJ

【 摘 要 】

The apoptotic protease-activating factor 1 (Apaf-1) split luciferase biosensor has been used as a biological tool for the detection of early stage of apoptosis. The effect of doxorubicin in a cell-based assay and the addition of cytochrome c and ATP in a cell-free system have been used to test the functionality of the reporter for the detection of apoptosome formation. Here, our data established a drug- and cytochrome c/ATP-independent way of apoptosis induction relying on the expression of the biosensor itself to induce formation of apoptosome. Overexpression of Apaf-1 constructs led to increased split luciferase activity and caspase-3 activity in the absence of any drug treatment. Caspase-3 activity was significantly inhibited when caspase-9DN was co-overexpressed, while the activity of the Apaf1 biosensor was significantly increased. Our results show that the Apaf-1 biosensor does not detect etoposide-induced apoptosis.

【 授权许可】

Unknown