Figure 1.
NTC efficiently kills mammalian cells and is compatible with other selection markers.
(A) HEK293T, HMEC, BT549, MDA-MB-468, U2OS and A2780 cells were plated (5000 cells/well) in 96 well plates and treated with indicated drugs 12 hours post plating. Cell viability was measured using MTS on the day the drugs were added (day 0), and 24 (day 1) and 48 hours (day 2) later. The MTS values were plotted relative to cell growth devoid of drug on day 2. (B) HMEC cells harboring selection markers for Neomycin, Hygromycin, Blasticidin and Puromycin (H-NPBH cells) were plated in 96 well plates. After 12 hours, the cells were treated with the indicated concentration of drugs. Cell viability was measured 72 hours post treatment using MTS. The percentage of growth is plotted as a function of drug concentration.
Figure 2.
Expression of NAT allows generation of stable cell lines expressing heterologous proteins using NTC.
(A) H-PNHB cells were infected with retroviruses encoding NAT and p100HA, and 12 hours later treated with NTC for selecting stable clones. Images of the infected cells, cells after 48 hours of selection and the generated stable cell line is shown. The black arrowheads indicate dying/dead cells. (B) Expression of HA-p36 in H-NH cells was induced for 24 hours with indicated amounts of Dox in the presence and absence of NTC. (C) Expression of HA-p100 in selected H-PNHB cells was induced with Doxycycline (Dox) for 24. Whole cell lystes were prepared after 24 hours and expression of HA-p100 was detected using an anti-HA antibody. Bands corresponding to HA-p36 and HA-p100 are indicated.