Can anyone suggest genetic and protein markers of necrotic cell death and autophagy in yeast S. cerevisiae?

As Caspase 3 assay is not applicable in S. cerevisiae, so what would be a good method to measure apoptosis/PCD in yeast cells?

We would be interested to have a simple method (maybe simpler than staining with tagged Annexin V?).

Thanks!

I am checking expression of autophagy -related proteins in yeast. To do so, I cultured and harvested cells in early stationary phase and very late stage (4 day culture). I planned to do western blot but I could not detect proteins on the short-lived strains I harvested at day 4 of culture using bradford assay. I prepared another lysate from a different sample of the same strain but detection is either 0 or close to zero in bradford. so I was wondering if the cells die (either apoptotic or necrotic), proteins can not be detected anymore. I am not actually sure if they actually died (just hypothetic since they are chronologically short-lived cells) though I senescent cells are also of possibility.

some article describes that more than one way of cell death (necrosis, autophagy, apoptosis) could possible in cells with single concentration of drug. How it is possible? how can single concentration of drug induce these types of cell death?

Hello everyone,

I want to see HMGB1 release to cell culture media to detect the necrosis levels. For this reason, I have used both lysates and cell culture media to make Western Blotting. But my media samples have smeared and have not run properly. You can see in the picture, the last three samples are media.

What do think about this problem? Is it because of the FBS? I thought that maybe I have too much HMGB1 but the first band of these three is the control group. So I do not expect any release in this sample but the view is the same.

Thank you in advance!

In a normal cell, which one of the below mentioned mechanisms of cell death occurs first?

Apoptosis or autophagy or necrosis

Autophagy (or autophagocytosis) (from the Greek auto-, "self" and phagein, "to eat"), is the basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components through the actions of lysosomes. The breakdown of cellular components promotes cellular survival during starvation by maintaining cellular energy levels. Autophagy allows the degradation and recycling of cellular components. During this process, targeted cytoplasmic constituents are isolated from the rest of the cell within a double-membraned vesicle known as an autophagosome. The autophagosome then fuses with a lysosome and its cargo is degraded and recycled.

There are three different forms of autophagy that are commonly described; macroautophagy, microautophagy and chaperone-mediated autophagy.

In the context of disease, autophagy has been seen as an adaptive response to stress which promotes survival, whereas in other cases it appears to promote cell death and morbidity.

I observe that a certain drug treatment induces autophagy in cancer cells. I am trying to distinguish the role of autophagy, to find out whether it aids in cell survival or autophagy activation leads to cell death (Type II programmed cell death). I am looking for short term experiments which could aid in this distinction. Any help in this regard would be greatly appreciated.

Hi everybody, I have a question in regard to apoptosis vs. necrosis.

I have cells from murine origin and I treated them with 2uM of Doxorubicin. This dose induces a massive apoptotic response and in fact, when I stain these cells with Annexin V I see an increased expression of this marker.

I also have the same cells exposed to 60C for 30 minutes (I am aiming to induce necrosis/cell death, perhaps different from apoptosis). When I stain the cells exposed to 60C they are positive for Annexin V and 7-AAD (a marker of cell death). However, the cells treated with Doxorubicin are Annexin V positive only (and 7-AAD negative). Therefore, I started to think that Doxorubicin is inducing an early apoptotic response and incubation of cells to 60C is inducing late/apoptosis necrosis. Based on this assumption, I collected the media from the cells exposed to the two different experimental conditions, I extracted the cfDNA and I ran a Bioanalyzer chip to see if I could observe any difference in the electropherogram.

Results: Cells Treated with Doxorubicin showed a majority of large DNA fragments. However, the cells exposed to high temperature showed a pattern rich of small fragments. I attach two examples here.

I am very confused because I was expecting small DNA fragments when apoptosis is involved and perhaps, longer DNA fragments when necrosis/cell death/late apoptosis is involved. This based on what is published in the literature.

Do you have any suggestion to explain this difference? Any insight would be greatly appreciated.

P.S. The Control (untreated cells) shows an electrophoretic pattern similar to the cells treated with Doxorubicin.