2.5. Plasmids and Cell Culture

U2OS human osteosarcoma cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS, Sigma, St. Louis, MO, USA) and 2 mM Ala–Gln (#G8541, Sigma). Mammalian expression plasmid, pRK5 β-catenin, has been previously reported [27]. pRK5 β-catenin variants were made by PCR oligonucleotide site-directed mutagenesis, as described [28]. Plasmids used for luciferase assays were pEZX-PG04 SEAP (GeneCopoeia, Rockville, MD, USA), pGL4.27 luc2P (#E8451; Promega, Madison, WI, USA), and pGL4.49 luc2P TCF/LEF (#E4611; Promega). U2OS cells were transfected using Effectene (Qiagen) transfection reagent following manufacturer’s recommendation.