I am generating mtDNA depleted cells using 100ng/ml EtBr and 50µg/ml Uridine. One which is p53 null, growing well even after 12 days of treatment but p53 positive one is showing completely different morphology as well as has vacuoles in cytoplasm. Any suggestions regarding this?
As for I know p53 has nothing to do with mitochondrial DNA. i had developed p0 cells using p53 null (H1299) and p53 positive cells (A549, MCF etc). Both are growing normally. Keep glucose and growth factor level optimun the in medium. I used low dose of EtBr (50 ng/ml) along with 2 mM pyruvate and 50 ug/ml uridine.
Hi,
As you are well aware that the metabolic pathways in genes are well connected. In case of p53 null cell line there may be some other compensatory mechanisms taking in action such as ATM or CHK kinases. Whereas, in case of functional p53, it is trying to repair the DNA damage, cuased by mutagens. vacuoles suggest that cells are undergoing apoptosis. It will be good for you to test the expression status of Cytochrome C (Mitochondrial Gene) and Other apoptotic genes such as p53, MDM2, ATM and CHK kinases.
Good Luck.
I suspect the other answers may reflect more expertise and experience than mine, but I believe that your results are likely informative as to the difference. P53 has many effects on stress responses that may impact mtDNA in my view. See for example - Vadrot N1, Ghanem S, Braut F, Gavrilescu L, Pilard N, Mansouri A, Moreau R, Reyl-Desmars F. Mitochondrial DNA maintenance is regulated in human hepatoma cells by glycogen synthase kinase 3β and p53 in response to tumor necrosis factor α. PLoS One. 2012;7(7):e40879. doi: 10.1371/journal.pone.0040879. It will be interesting to see what you find out.
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