hi christiana! the only suggestion I have is that the kinetics f PARP cleavage is different. DNA-fragmentation is a late stage readout....i observe somethimes only partial PAPR cleavage in time-course experiments, sometimes the full-lenght PARP is not disappearing...just a reminder: also harvest the floating,detached cells from the supernatant...good luck!

1. If my guess is correct, MCF7 should have no PARP cleavage because they are caspase-3 deficient. However, there could be varient MCF7 cells out there in various laboratories.

2. When you use z-VAD-fmk with apoptosis inducing stimuli such as your synthetic compound, the cells will shift the balance from apoptosis to necrosis (due to RIPK) and when necosis occurs there wont be DNA fragmentation but still PARP will be cleaved by granzyme-B/lysosomal proteases. In such occasions you'll see a 42kDa PARP cleavage (along with approx 80kDa band; 116-42=74)

3. Only with certain antibodies you can see the 42kDa PARP bands because these days the antibodies are raised to see only cleaved PARP (89kDa) and hence some antibodies may not detect 42 and 74 kDa bands. See this paper for more details and antibodies that can detect full length+42+89kDa PARP bands (PS: this antibody described in this paper is good to use in other cell lines and the RT4P cell line is exceptional and has multiple bands) https://www.researchgate.net/publication/233742776_Blebbishields_the_Emergency_Program_for_Cancer_Stem_Cells_Sphere_Formation_and_Tumorigenesis_after_Apoptosis?ev=prf_pub

Article Blebbishields, the Emergency Program for Cancer Stem Cells: ...

Hi Christiana. Casp-7 can cleave PARP as well, so you will see this happening also in MCF-7 cells. I think the very first papers on PARP cleavage actually link it to caspase-8 (which indeed has some activity against PARP as well).

In your case (MCF-7 vs MDA cells) it might well be that the molar amounts of zVAD targets (i.e. caspases) as well as their natural inhibitors (i.e. XIAP) and antagonists of these inhibitors (i.e. Smac) could be very different. Even without casp-3, MCF-7 could therefore potentially withstand higher amounts of zVAD (what conc. do you use?).

I'm pretty sure that MCF-7 do not express RIP3, so necroptosis is not that likely as an explanation. Also, I suppose your compound induces the intrinsic pathway?

Hi, thanks for your answer. I have checked for Caspase-3,-2,-9,-8,-7 and -6 cleavage at several time points but I've only been able to detect the 28 kDA fragment of Caspase-7 at 12 hours in MCF-7 cells which then disappears. I have used z.vad up to 20 μΜ-after that it gets toxic. I also checked for necroptosis using Necrostatin but there was no rescue in viability. On the bright side, I do have Survivin downregulation in MCF-7 and MDA cells that may account for the evidence of caspase activity without an increase of the cleavage product-supposing that active caspases are released from IAPs-as well as AIF and Endo G release in MCF-7 cells only. My concern is that in MDA cells I do have restoration of DNA fragmentation with z.vad.fmk but not PARP cleavage. My question is: is it possible that ICAD is activated by either a Caspase that does not cleave PARP or by Calpains or Cathepsins directly?

I suppose a big contribution towards what you can see/detect or not is also the antibody quality, the target abundance, and the % of cells responding to treatment. In that sense, it is difficult to judge without seeing the data whether there really are substantial differences between your cell lines. We use zVAD at 50 uM to fully block apoptosis execution in MCF-7, by the way. In some cell lines you need to go even higher. These zVAD concentrations block caspases, but the cells (if going through the intrinsic pathway) will stll die by necrosis due to the bioenergetic crisis following MOMP. Also, you will notice in proliferation assays, that even small amounts of zVAD impair proliferation, so the viability assay you use may be affected by that. Did you measure cell death (e.g. by propidiumiodide uptake or similar)? Are cell death amounts (%) similar in both cell lines and does zVAD protect both cell lines?

I used up to 40 μΜ z.vad and saw a 15% decrease in the viability of MCF-7 cells with the MTT assay so I avoided going over 20 μΜ. But you are right, higher z.vad concentration will possibly lead to complete restoration of DNA fragmentation in MCF-7 cells as well. In the MTT assay I also did not observe a rescue of cell viability when I co-incubate the compound with z.vad which means that the cells still die via a caspase-independent pathway. I also observed an increase of the SubG1 fragment after exposure to this compound-which was greater in MDA cells-but co-incubation with z.vad produced inconclusive results. I should probably repeat this :) Thanks for all your help.