This happened to my 3T3 cells and I had to take them off of the antibiotic. You could have kept the cells in too high a concentration G418, which will result in cytotoxicity and cellular stress. Have you established a kill curve for your cells with G418?

First generate a kill curve which is highly recommended before establishing stable cell line; which is a dose responsive experiment where u treat your cells with an increasing amounts of antibiotic to determine the minimum antibiotic conc that can kill your all cells over the course of one week.

In my experiences, massive cell death at a certain time point seems to be a natural phenomenon when generating stable cells; probably this point would be when transiently expressed resistence gene products have gone and only the cells stably integrated the resistence gene survive and begin to form colonies. Cell death is massive because the chance of random integration is quite low.

I agree with Dooyoung Lee. When you are trying to establish a stable cell line, only the stably transfected cells will survive the introduction of antibiotic. The stable integration efficiency is typically low so you will see a massive die off of those cells which are not transformed. In my experience, it takes about 3 weeks to start seeing healthy foci after the introduction of the antibiotic. At that point you can select for monoclonal clones or combine all the foci for a polyclonal line (if you are using random integration).

Amended on 12 March 2018

I have experienced in meningitis that two phase increase of biotinidase, which seems to be strongly linked to immunity, in cerebrospinal fluid (CSF) has been occurred (please see file; meningitis BIN). Your DNA fragment , which mimics DNA virus, may have induced ovarian cancer cells to produce immunity or defending proteins. Ovarian cancer cells may have HIV-1 and a +ssRNA virus (possibly HAV) in latent state, and they may be induced by your DNA fragments' stress. HIV-1 and HAV may also induce breast cancer and cervical cancer, since HAV seems to like human female (our unpublished observation). Both induced immunity and induced virulent virus may have induced your cell-death phenomenon after 6 days.

By the way, I have searched POTE ankyrin domain family member B/ Prostate, Ovary, Testis Expressed ankyrin domain family member B/Prostate, ovary, testis-expressed protein on chromosome 15/ANKRD26-like family B member 1 in vain (please see file; HepG2 fucoidan).

Then, I have searched ANK and ANKRD proteins.

Hepatoma HepG2 (cultured without fucoidan) has Ankyrin-2/Brain ankyrin at 0.7, Ankyrin repeat and fibronectin type-III domain-containing protein 1/ANKFN1/FANK1 at 2.0, Ankyrin repeat domain-containing protein 15/KANK1/KN motif and ankyrin repeat domain-containing protein 1 at 0.83, and Ankyrin repeat domain-containing protein 50/ANKRD50 at 5.0 μg/mg of cell protein, respectively (Total 8.5 μg/mg of cell protein).

Healed normal hepatocyte HepG2 (cultured with fucoidan) has Ankyrin-2/Brain ankyrin at 1.5, Ankyrin-3/ANK-3 at 1.4, Ankyrin repeat and fibronectin type-III domain-containing protein 1/ANKFN1 at 1.7, Ankyrin repeat domain-containing protein 38/KN motif and ankyrin repeat domain-containing protein 4 at 2.5 μg/mg of cell protein, respectively (Total 7.1 μg/mg of cell protein).

LC tissue (named as No.6) has Ankyrin-1/ANK-1/Ankyrin-R/Erythrocyte Ankyrin at 6.9 μg/mg of tissue protein, but HCC tissue (No.6) has it c.a. 5-fold lower amount of 1.4 μg/mg of tissue protein.

Interestingly, the expression of ANK and ANKRD proteins seems to be up-regulated by HCV and competitively down-regulated by HAV, Japanese encephalitis virus (JEV), Turnip yellow mosaic virus (TYMV), Bean yellow mosaic virus (BYMV), Hantaanvirus (HTNV), and Enteric Cytopathic Human Orphan virus (ECHO virus). Then, although the effect of TYMV and BYMV on humans has not been clarified, ANK and ANKRD ruled by these virus seem to be linked to membrane-related diseases in brain and kidney.

Healthy serum (52y, female) has Ankyrin repeat domain-containing protein 50/ANKRD50 at 4.0 μg/mg of serum protein.

Healthy serum (22y, female) has Ankyrin repeat and KH domain-containing protein 1/HIV-1 Vpr-binding ankyrin repeat protein at 2.8 μg/mg of serum protein.

Interestingly, exclusion of these proteins into blood seems to be induced by Mycoplasma pneumoniae, although correlation of serum protein and organ protein has not yet been clarified.

Therefore, ANK and ANKRD proteins seem not to be linked directly to the cancer, but they are required for normal cells to alive.