In order to explore the role of Smac/DIABLO in H2O 2-induced apoptosis of C2C12 myogenic cells, Hoechst 33258 staining was used to examine cell morphological changes and to calculate percentage of apoptotic nuclei. DNA ladder pattern on agarose gel electrophoresis was used to observe DNA fragmentation. The release of Smac/DIABLO from mitochondria to cytoplasm was observed by Western blotting. Activities of Caspase-3, Caspase-9 were assayed with Caspase Colorimetric Assay Kit and Western blotting. Full length Smac/DIABLO gene was transiently transfected in C2C12 myogenic cells by lipofectamine and then protein levels of Smac/DIABLO were analysed by Western blotting. The results showed that; (1) H2O2 (O. 5 mmol/L) resulted in a marked release of Smac/DIABLO from mitochondrial to cytoplasm l h after treatment, activation of Caspase-3, Caspase-9 4 h after treatment and specific morphological changes of apoptosis 24 h after treatment; (2) Smac/DIABLO overexpression significantly enhanced H2O2 induced apoptosis in C2C12 myogenic cells as shown by specific DNA ladder pattern in agarose gel electrophoresis, increase of percentage of apoptotic nuclei and marked activation of Caspase-3, Caspase-9. These data suggested that H2O2 could result in apoptosis of C 2C12 myogenic cells, and that release of Smac/DIABLO from mitochondrial to cytoplasm and the subsequent activation of Caspase-9 and Caspase-3 played important roles in H2O2-induced apoptosis in C2C12 myogenic cells.
|Original language||English (US)|
|Number of pages||7|
|Journal||Progress in Biochemistry and Biophysics|
|State||Published - Dec 1 2003|
- CC myogenic cells
- Hydrogen peroxide
ASJC Scopus subject areas