Is it possible that a KD cells showed reduced cell proliferation by WST but during FACS analysis it did not show any change of cell cycle? apoptosis checking by cleaved caspase 3 western blot also no significant change. So what happened?
Hi Sya,
I hold the view that you are one of proficient scientist, so I am pleased to discuss this issue with you.
I am curious if you have ever investigate the level of cell division marker. The accumulated cell cycle (cell growth) causes cell division and we call it as cell proliferation.
So many factors are concerned in cell cycle, like growth factor, proliferating complexes, or division enzymes. And separase, regulating division of cell, could be one of critical factors.
However, as we know, if the level of separase is downregulated, the imbalnced DNA quantity cause the instability of viable cell. Nevertheless, it is critical, the cell cycle is not affected by absence of separase.
This events could be proved by confirming number of chromosome in a single cell.
However I cannot guess the reason why the level of separase in KD mouse influenced by WST (water-soluble tetrazolium salt).
My assumption could be wrong, and not be important in your work procedure, but I offer my opinion cautiously.
I'll give you a reference :
Article The selective continued linkage of centromeres from mitosis ...
And here are some excerpts from the article.
"failure of chromosomal segregation is not the cause of cell death in separase-deficient cells."
"the presence of nonsegregated chromosomes in separase-deficient cells does not appear to cause additional defects in cell cycle progression events, such as DNA replication or centrosome duplication."
Thank you.
Junhyung.
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