Coon s modified ham s f12 medium

FRT (Fisher rat thyroid cells) cells were cultured in Coon’s Modified Ham’s F12 Medium (Euroclone, Milan, Italy) supplemented with 5% foetal bovine serum (Euroclone), 1% penicillin (Euroclone) and 1% streptomycin (Euroclone). U937 cells were cultured in RPMI supplemented with 10% foetal bovine serum. FRT cells were stably transfected with CET 1019 AS-Puro-SceI- FOXE1 plasmid using FuGene 6 Transfection Reagent (Roche, Basel, Switzerland) according to manufacturer’s instruction. Briefly, 3 μg of CET 1019 AS-Puro-SceI-FOXE1 plasmid or 3 μg of empty vector was transfected in FRT cells seeded in 100 mm culture dishes (Corning, NY, USA) at 20% confluence. Forty-eight hours after transfection, cells were selected in complete medium supplemented with 1 μg/mL of puromycin. After two weeks of continuous selection, single colonies were picked from plates transfected with either CET 1019 AS-Puro-SceI-FOXE1 vector or empty vector. The clones overexpressing the greatest amount of FOXE1 (FOXE1-FRT) were used for the showed experiments.

The use of human fetal tissue for research purposes was approved by the National Ethics Committee and the local ethic committee for investigation in Humans of the University of Florence (Permit Number: 678304). Human fetuses biopsies were obtained from therapeutic medical abortions after women approved and signed the informed consent document, as already reported (Gallina et al., 2008 (link)). NBM tissue was dissected from two female 12-weeks old human fetuses and incubated with 1 mg/ml collagenase type IV (Sigma-Aldrich Corp., St. Louis, MO, USA). The cell suspensions were mechanically dispersed by pipetting and cultured in Coon’s modified Ham’s F12 medium (Euroclone, Milan, Italy) supplemented with 10% FBS (Hyclone, Logan, UT, USA). Cells were used within the 26th passage.