3 replies to this topic

[cancerous]

Hi all,

I am studying apoptosis whch is induced by drugs or serum starvation.

I found that PARP is cleaved by these apoptotic stimuli in breast cancer cells but this cleavage peaked at certain time point and the cleaved form decreases. Please see my western blot image.

I assumed that PARP cleavage would keep increasing during apoptosis process unless cells are completely dead and proteins start to be degraded. However, in my western blot, PARP cleavage peaked at 24hr starvation and went down at 72hr. When I harvested the cells at 72hr, cells did not look completely dead because they are all attached to the bottom and morphology was not changed. Also, equal loading indicates that there is no significant protein degradation is going on at 72hr starvation (or cleaved PARP is extremely labile??).

Could anyone explain why PARP cleavage decreases after peaked during apoptosis? Thanks!

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Edited by cancerous, 07 March 2012 - 06:08 PM.

[Curtis]

in my case, during apoptosis only some of my cells died and the rest continued proloferating. the division time for human cells is normally 24 hr. It is possible that your healthy cells are still dividing and you are collecting sister cells. As the result the PARP cleavage shows decrease in western blot.

So I collected my ' floating dead cells ' separately from the attached cells. I don't know why not so many people practice this.

In late-apoptotic cells PARP cleavage does not dissapear. You must still see PARP cleavage in western blot.

in early-apoptotic cells (which most of them are still attached to the flask) you must see less PARP cleavage.

How do you starve them anyway?

Edited by Curtis, 16 March 2012 - 07:41 PM.

[cancerous]

Hi Curtis,

Thank you for your opinion. I first wash cells twice with PBS, and then add serum-free medium. You may be right in that live cells still are proliferating and total PARP cleavage is just diluted out. However, I also treated drugs such as staurosporine to induce apoptosis and observed incease in PARP cleavage for some time and the cleaved PARP decreased later. In this case, there is no live proliferating cells. Then why are cleaved PARP levels decreasing?

[Curtis]

sorry for late reply. staurosporine induces intrinsic pathway....so PARP cleavage must remain strong until the end. I'm not sure why this is happening to you. can you check other antibodies too? Caspase 3 or cytochrome c?