FEAT overexpression in NIH3T3

To evaluate whether FEAT has cellular functions relevant in oncogenesis, FEAT was overexpressed in NIH3T3 cells, which only weakly express FEAT protein, and the alterations in genome-wide transcriptional profiles were analyzed by microarrays. The ORF of human FEAT cDNA was subcloned into the pENTR3C entry vector of the Gateway cloning system (Invitrogen). LR recombination with the pEF-DEST51 vector (Invitrogen) yielded the pEF-DEST51-FEAT plasmid that encodes FEAT driven by the elongation factor 1alpha promoter. NIH3T3 cells were transfected with pEF-DEST51-FEAT using HilyMax (Dojindo Laboratories). After 7 days of selection with 10 µg/ml blasticidin S (Kaken Pharmaceutical), colonies were picked and screened for clones in which FEAT protein was overexpressed. Three clones, FEAT-3, -15, and -20 were used in further studies. The ccdB gene in the pEF-DEST51 vector was deleted to construct the pEF-DEST51-∆ccdB plasmid, which was stably transfected into NIH3T3 cells to obtain control cell lines, named ∆ccdB-1, -2, and -3.