Optimization of Experimental Variables Influencing Apoptosome Biosensor in HEK293T Cells
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 <i>c</i> and ATP in a cell-free system have been...
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| Format: | Article |
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MDPI AG
2020-03-01
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| Series: | Sensors |
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| Online Access: | https://www.mdpi.com/1424-8220/20/6/1782 |
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| author | Azarakhsh Oladzad Maryam Nikkhah Saman Hosseinkhani |
| author_facet | Azarakhsh Oladzad Maryam Nikkhah Saman Hosseinkhani |
| author_sort | Azarakhsh Oladzad |
| collection | DOAJ |
| description | 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 <i>c</i> 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 <i>c</i>/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. |
| first_indexed | 2024-04-11T21:47:00Z |
| format | Article |
| id | doaj.art-ac0460e1a3d849669255e16b02184c81 |
| institution | Directory Open Access Journal |
| issn | 1424-8220 |
| language | English |
| last_indexed | 2024-04-11T21:47:00Z |
| publishDate | 2020-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Sensors |
| spelling | doaj.art-ac0460e1a3d849669255e16b02184c812022-12-22T04:01:23ZengMDPI AGSensors1424-82202020-03-01206178210.3390/s20061782s20061782Optimization of Experimental Variables Influencing Apoptosome Biosensor in HEK293T CellsAzarakhsh Oladzad0Maryam Nikkhah1Saman Hosseinkhani2Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran P.O. Box 14115-175, IranDepartment of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran P.O. Box 14115-175, IranDepartment of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran P.O. Box 14115-175, IranThe 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 <i>c</i> 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 <i>c</i>/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.https://www.mdpi.com/1424-8220/20/6/1782apoptosisapoptosomeluciferase complementary assay |
| spellingShingle | Azarakhsh Oladzad Maryam Nikkhah Saman Hosseinkhani Optimization of Experimental Variables Influencing Apoptosome Biosensor in HEK293T Cells Sensors apoptosis apoptosome luciferase complementary assay |
| title | Optimization of Experimental Variables Influencing Apoptosome Biosensor in HEK293T Cells |
| title_full | Optimization of Experimental Variables Influencing Apoptosome Biosensor in HEK293T Cells |
| title_fullStr | Optimization of Experimental Variables Influencing Apoptosome Biosensor in HEK293T Cells |
| title_full_unstemmed | Optimization of Experimental Variables Influencing Apoptosome Biosensor in HEK293T Cells |
| title_short | Optimization of Experimental Variables Influencing Apoptosome Biosensor in HEK293T Cells |
| title_sort | optimization of experimental variables influencing apoptosome biosensor in hek293t cells |
| topic | apoptosis apoptosome luciferase complementary assay |
| url | https://www.mdpi.com/1424-8220/20/6/1782 |
| work_keys_str_mv | AT azarakhsholadzad optimizationofexperimentalvariablesinfluencingapoptosomebiosensorinhek293tcells AT maryamnikkhah optimizationofexperimentalvariablesinfluencingapoptosomebiosensorinhek293tcells AT samanhosseinkhani optimizationofexperimentalvariablesinfluencingapoptosomebiosensorinhek293tcells |