Hi Pasupathi,

What is the percentage of apoptotic cells you observed in Annexin staining? I mean Annexin positive %, PI positive % and double positive %.

PARP was disappeared very interesting. Did you probe the blot for any loading controls like GAPDH, Tubulin or Actin? 

Please go through the attached article to probe for other forms of cell death.

Good luck

Ram

Dr.Ramakrishnan Gopalakrishnan,

Thank you for your reply. I have attached my data for your reference.

I could not find any cleavage of caspase-3 and PARP even over exposer also.

As you suggested I will read that article. mean while if you have any suggestion with my data, kidly let me know.

Thanks in Advance.

If your cells are still dying in presence of a pan-caspase inhibitor (QVD or zVAD-fmk) this is CICD.

Your right, I agree with you. still, I think without cleavage of Caspase and PARP,  it would be waste of time and money trying pan-caspase inhibitor.  

Hi Pasupathi,

Yeah i agree your data is very clean. I just have one more question Can you please check with the datasheet of the antibodies for Caspase-3 and PARP whether they can detect the cleaved Caspase-3 and cleaved PARP.

1. Some antibodies only detect the full length proteins not the cleaved ones. That's why i am just wondering whether your antibody can detect only the full length.

2. Looking at your PARP blot  it looks like it is cleaving the PARP and the cleaved PARP runs around 85 kda.

3. I used this antibodies from PARP (9542)  from cell signalling which detects both the full length and cleaved PARP and i used this cleaved Caspase-3 (9664) antibody from cell signalling. For cleaved caspase-3 you may have to expose your blot for longer time may be hours to detect the cleaved. Please see the attached article for more information.

Good luck

Ram

Dr.Ramakrishnan Gopalakrishnan,

your right, I am using same antibodies only.

1. In case PARP, I am 100% sure, I colud not detect cleaved PARP( I tried so many times and I have cleaved PARP antibody also from cell signalling)

2. Coming to cleaved Caspase-3, I did not exposer such long time. As you suggested I will try. but I used Pro-caspase-3 it can also detect cleaved cas-3 (antibody from cell signalling -9662 (37, 19, 17 kda).

3. Finally its hard to prove that my compound induced apoptosis. I hope like you experienced person can help me to overcome from this issue?

Hi Pasupathi,

If you don't see cleaved PARP or Caspase-3 that is fine. Before venturing into capase independent cell death or other types of cell death first i will check why your compound drastically degrades the level of PARP.  Because looking at your results the first thing that struck me is the enormous downregulation of PARP by your compound.  Going further into that this is what i will do.

1. Check whether the mRNA level of PARP is also going down. If yes then the downregulation is at the transcriptional level.

2. If not then i will treat the cells with your compound in the presence of cycloheximide (protein synthesis inhibitor) to check whether your compound directly degrades the PARP protein.

Let me know what's happening.

Ram

Dr.Ramakrishnan Gopalakrishnan,

Absolutely your right, I have an answer for this also.

Already I checked mRNA levels also, there is no change.

1. why you suggest cycloheximide, I did nt understand?

2. cycloheximide is know to induce PARP cleavage, my compound make it disappearance of PARP

3. Combination treatment what will happen?

4. I think my compound will be more active than cycloheximide.

5. one more thing, i would like to share with you, it will be critical, still this is the fact, my compound down regulating p53 as well.

Its hard to make any new experiemnt and conlusion.

I am really happy to discuss with you, I have learned many things form you.

Thanking you,

Pasupathi.

Annexin V staining can also occur if the plasma membrane has been permeabilized. Did you counter-stain with propidium iodide? If the cells are Annexin V positive and PI negative, then they are apoptotic.

Hi Adrienne Gorman,

Thank you for your suggestion. i did counter-stain also. I hope , If you see my data, you will uderstand. My concern is how can i prove apoptosis by western blot?

Hi Pasupathi,

You already checked mRNA and there is no change. Then here is the answer, Your drug works post-translationally on PARP by decreasing the protein half-life/stability. To confirm the above point you need to do the cycloheximide experiment. I didn't tell you to do a combination experiment. Please see Fig 4B of my paper attached earlier in which I have done this cycloheximide chase experiment to determine the half-life of protein. Briefly, Cycloheximide (CHX) is a protein synthesis inhibitor. Then if you treat the cells with and without drug which will give you the information whether your drug degrades the PARP faster than normal. If yes then it is possible that your drug works by destabilizing PARP. And it is no secret that many PARP inhibitors are effective against breast cancers. If your compound rapidly downregulates p53 also you can strip the same blot (CHX experiment PARP blot) and probe for p53 also. Remember different protein have different half-lives so you may have search for earlier publications to choose optimum time point for your experiment. Just curious why are you using mM concentrations of your drug it works only @ such higher concentration?

Good luck!

Ram

Check other capsase dependent proteins as Fodrin (alpha-spectrin) by wester blot... annexin-v mostly indicated necrosis rather than apoptosis.... however, the caspase kit assay may help as well.