| Year | 2011 |
|---|---|
| Journal Info. | Molecular & Cellular Toxicology, 7, 177-184 (2011). |
| Author | Kyoung Hwa Jung*, Young Me Song*, Nando Dulal Das, Kyoung Sun Park, Mi Ran Choi, Sang Youn Hwang, Eun Kyu Lee, Moon Kwon Lee, Jaebum Choo, Kyoung Suk Kim, Moo Soung Kim, Sang Rin Lee, Young Gyu Chai |
| etc. |
Abstract
Caspase-3 is a key mediator of apoptosis in mammalian cells. Cells expressing caspase-3 substrate peptides have become powerful and increasingly common components of cell-based assay systems. We developed a cell-based assay to measure staurosporine (STP)-induced caspase-3 activity in live rat glioma C6 cells. The caspase-3 sensing system was constructed to include a nuclear export signal (NES), followed by the amino acid sequence Asp-Glu-Val-Asp (DEVD) and a green fluorescent protein (GFP) fused to the Nterminal site of a nuclear localization signal (NLS). Using time-lapse confocal microscopy, we monitored caspase-3 activation during apoptosis by imaging the translocation of GFP from the cytosol to the nucleus. After 8 h of 0.5 μM STP treatment, caspase-3 activity was assessed by monitoring the translocation of GFP to the nucleus due to cleavage of the NES from the GFP by caspase-3. Finally, disintegration of the plasma membrane during late apoptosis was confirmed using a nuclear dye, propidium iodide. Analysis of caspase-3 activity using real-time monitoring can potentially be used to screen for apoptotic molecules in living cells.
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