Insuline

Human Embryonic Kidney (HEK293) and Human cervical carcinoma (HeLa) cells were cultured in DMEM 10% fetal bovine serum (FBS). Murine inner medullary collecting duct (IMCD3) cells were cultured in DMEM-F12, 10% Fetal Calf Serum for RNA in situ hybridization experiments. Human Kidney 2 (HK2) cell were cultured in D-MEM/D-MEM F12 (1:1) 5% FBS supplied with 1% Glutamine and ITS (Insuline 5 ug/ml, Transferrine 5 ug/ml and Selenium 5 ng/ml) from SIGMA.
Media were supplemented with 100 Units/ml penicillin, and 100 μg/ml streptomycin. Cells were grown at 37 °C with 5% CO₂.
Cycloheximide (CHX) (C-7698, SIGMA) and MG132 (C221, SIGMA) were used at 100 µM and 30 µM concentration, respectively, to treat cells for 6 hours.

Tumor sphere cultures were established from trypsinized cells from adherent cultures of the parental OE33 and OE19 cell lines. For the formation of esophageal spheres, cells were plated in 25 cm² ultra-low attachment flasks (Corning, Tewksbury, MA, USA) and cultured in serum-free Dulbecco's modified eagle medium (DMEM-F12, Sigma) containing: 5 μg/mL insuline (Sigma), 0.4% bovine serum albumin (Sigma), 10 ng/mL human basic fibroblast growth factor (bFGF; Sigma), 20 ng/mL epidermal growth factor (EGF; Sigma), 2% B27 Supplement (Invitrogen Corporation, Grand Island, NY) and antibiotic/antimycotic (Sigma). Cells were supplemented with fresh medium and growth factors twice weekly. At day 14, spheres >40 μm in diameter were collected using a cell strainer (BD), dissociated to single cells, and reseeded to evaluate self-renewal through the formation of a second generation of tumor spheres. In order to determinate putative CSC markers, an aliquot was analyzed by flow cytometry.
For differentiation assays, disaggregated spheres were seeded in RPMI 1640 medium with 10% FCS in adherent conditions.
In another set of experiments, OE33 cells were plated for the formation of esophageal spheres and then treated with celecoxib (10, 20, or 40 μM) for 7 days. Thus, we determined the size of spheres using Image J software at days 2 and 7 by measuring sphere diameter.

First-passage ASCs were replated on thermanox cover slips (Nunc, Roskilde, Denmark) for adipogenic differentiation, and another part for osteogenic differentiation. Adipogenic differentiation was performed using basic culture medium with addition of dexamethasone, insuline, indomethacine and 3-isobutyl-1-methylxanthine (Sigma-Aldrich, St Louis, MO, USA). Differentiation was confirmed by Oil Red O stain. Osteogenic differentiation was performed using basic culture medium with addition of dexamethasone, ascorbic acid and β-glycerophosphate (Sigma-Aldrich, St Louis, MO, USA). Differentiation was confirmed by Von Kossa's Stain. Digital imaging of all wells was performed using an Olympus inverted microscope, and Cell^M software (Olympus Europe, Hamburg, Germany).