Skip to main content
. Author manuscript; available in PMC: 2015 Nov 21.
Published in final edited form as: Oncogene. 2014 Jul 21;34(21):2807–2813. doi: 10.1038/onc.2014.211

Figure 1.

Figure 1

Nfkb1 prevents alkylator-induced mutation. (a) Hprt mutation assay in primary Nfkb1+/+ and Nfkb1−/− MEFs treated with increasing concentrations of TMZ and selected in 5 µg/ml 6-TG. Data show mean number of 6-TG resistant (TGr) clones ± SEM of 4 independent experiments performed at least in duplicate. * P < 0.05. (b) Hprt assay in Nfkb1−/− MEFs stably expressing p50 or empty vector (EV) following treatment with TMZ. * P < 0.05. (c) Alkaline comet assay in Nfkb1+/+ and Nfkb1−/− MEFs treated with vehicle (UTC) or 100 µM TMZ for 3 hours. Graph demonstrates quantification of average tail length normalized to nucleus diameter ± SD. * P < 1×10−5. N = 50 cells. Experiment was repeated. Hprt assay in (a) was performed using early passage primary MEFs, harvested from day 14 embryos, treated with TMZ and maintained in exponential growth for 7 days. Cells were subcultured in growth medium alone (plating efficiency) or in the presence of 5 µg/ml 6-TG for selection of mutants. Induced Hprt mutations were calculated as the number of 6-TG resistant colonies per 106 cells plated after correction for plating efficiency. Importantly, 5 ug/ml 6-TG is 100 % lethal to un-mutated MEFs of both genotypes. For re-expression of p50, immortal Nfkb1−/− MEFs were infected with the retroviral vector MigR1-p50 or MigR1-EV. MigR1-p50 was created by liberating p50 from pcDNA3.1-p5033 by digestion with PmeI and XhoI, and ligated into the BglII site of pMSCV-MigR1 containing an IRESGFP insert. Retrovirus was produced with Platinum-GP packaging cells following transfection of MigR1-p50 or MigR1-EV using XtremeGENE transfection reagent (Roche). MEFs were then spinoculated with virus/polybrene-containing supernatant and colonies sorted by FACS. Alkaline single cell gel electrophoresis (Comet assay) was performed as described.34 Briefly, MEFs were treated with TMZ for 3 hours, trypsinized and resuspended in low-melting point agarose. A single cell suspension in the agarose was layered on glass slides and following cell lysis, submerged in alkaline (pH > 13) buffer and electrophoresis performed until slight migration pattern observed in the untreated nuclei. Dried slides were stained with ethidium bromide and visualized by fluorescence microscopy. Tail length was quantified as tail length normalized to nuclear diameter.