Dear Ewelina Honkisz ,

The pictures are quite nice, of suffienct quality to be used for further analysis. What is a pity, is that the treated cells are not represented on the same scale, magnofication as the treated cells.  However, I think I can tell them apart from each other by the complicated vacuolar structures in the treated cells vs the controls. 

The first 2 images (page 1) show a cell, with part of the nucleus, and a clear nucleolus.

The same for the second image (page 2), but without a nucleolus.The vacuole is simple spheric in its structure, with embedded old vacuolar remains.

Page 3 is a problem for me.It is labelled as control,but based on my interpretation of the image I would classify it as a treatment. It shows excessive membrane extensions, numerous small vacuoles and a complicated outher cell membrane. Also a lot of pycnotic mitochondria. Does not seem to be compatible with the images from page 1 and 2.

Page 4 and 5 are at different magnifications than the first 3 pages, without an overview of the entire cell.But they show a lot of vacuolar inavaginations, pycnotic mitochondria.

I hope this may be helpful. Kind regards, Harrie Verhoeven.

Dear Ewelina,

I agree with Harrie. In page 3 the structures look like bona-fide autophagosomes. In the later images the structures appear much larger.

If you consider doing a quantification it would be best to do it blind without knowing which sample has been treated. You could normalize the number of autophagosomes or their size to the cytoplasmic area.

Kind regards,

Andreas

Harrie A Verhoeven and Andreas Müller  thank You very much for your answer. For sure if I understood you correctly, the picture with treated cells (4, 5 and 3 even if it is signed "control" - this might be a mistake of my colleague) have a halmark of autophagy. If am I correct, then what is on the picture 2 - double-membrane structere inside vesicle?

Dear Ewelina,

the double membrane is the hallmark of autophagy. The structure containing the double-membrane object might be an autolysome. It's sometimes hard to tell what an object is when you see additional membranes (more than the double membrane). Also the large vesicles in the treated images might be autolysosomes. When you induce autophagy you usually increase the degradative pathway. So you might see many more lysosomes and autolysomes since autophagy is just the mediating pathway of intracellular degradation. Ultimately, some CLEM might be interesting here. Or immuno-EM.

I hope this helps,

Andreas