Glycyl histidyl lysine

The differentiated strain of rat-thyroid cells (Fisher-rat-thyroid-line-5; FRTL5; American Type Culture Collection, Manassas, VA; ATCC CRL 8305, F1 subclone) served as an in vitro experimental substrate. Cells were grown for 1 week in 6H medium (Coon’s Modified Ham’s F-12 Medium (Sigma Chemical Co.) supplemented with 5% adult calf serum (BioWhittaker, Inc., Walkersville, MD) and a six-hormone mixture containing insulin, somatostatin, hydrocortisone, transferrin, glycyl-histidyl-lysine, and TSH (Sigma Chemical Co.).

Thyroid carcinoma cells FRO [15] (link) available in our laboratory and HEK 293 cells (American Type Culture Collection, LGC Standards S.r.l., Sesto San Giovanni, Italy) were grown in DMEM (Life Technologies, Grand Island, NY) supplemented with 10% fetal calf serum (Life Technologies), 1% glutamine, 1% penicillin/streptomycin (Life Technologies) in a 5% CO₂ atmosphere. FRO and HEK 293 cells were transfected using either Lipofectamine reagent (Invitrogen, Carlsbad, CA) and Neon Electroporation System (Invitrogen) according to manufacturer's instructions. To generate CBX7 stable expressing cells, transfected cells were selected in a medium containing geneticin (Life Technologies), several clones were picked and expanded for further analyses. Rat normal thyroid PC Cl3 cells [16] (link) were cultured in modified F12 medium supplemented with 5% calf serum (Life Technologies) and six growth factors (thyrotropic hormone, hydrocortisone, insulin, transferrin, somatostatin and glycyl-histidyl-lysine; Sigma, St. Louis, MO). PC Cl3 cells were transfected with control siRNA and siRNA against rat cbx7 as previously reported [11] (link). Mouse embryonic fibroblasts (MEFs) from cbx7 knockout mice were established, grown and transfected (passage P3 and P4) as described elsewhere [10] (link).