The inhibitors that you tried are cytotoxic. Keratinocytes are rounded before they died. 

Dear Nur,

You must be referring to vacuolization of the cells, when you say formation of balloons, which is a sign of necrosis.

best wishes, Refik

Hi Nur Mahammad,

The followings are the main types of cell death related morphological changes:

(1)   Necrosis: appearance of membrane bubbles, cell swelling, disrupted cell membranes.

(2)   Apoptosis: cell rounding and shrinkage, blebbing/apoptotic bodies, nuclear condensation

(3)    Autophagy: extensive vacuolization, autophagosomes formation.  

From the observations that you have shared so far, the cells in your experiments seems be going through necro-apoptosis but it would be great if you can manage to send an image of the cells. For the molecular mechanisms involved in cell death, you can look at the Apoptosis Poster at http://www.novusbio.com/mailing-list

As far as your further experiments with these inhibitors are concerned, I would suggest dose/time optimization by trying a relatively lower concentration of the inhibitors and a couple of different time points also.

Best Wishes

Subhash

Regarding to HaCaT cells described picture is common and share apoptotic features more than necrotic. We can see floating "completely round balloon" with attached condensed cellular debris in our experiments too.

In our experience UV-B treated HaCaT also demonstrated non-typical apoptosis picture. Some autophagy sings were found 6 hr after UV exposure and very small increase in Anexin V + and PI + cell number. After 24 hr we were able to detect twice or more increased level of apoptotic cells (AnV+PI+-) but it was related to mitotic catastrophe during cytokinesis.

So, taking in account problems with p53 in this cell line and corresponding problems with induction of apoptosis these keratinocytes are NOT good model for normal keratinocytes. Nevertheless they are able for form histologically normal skin in some cases.