Ham s f12 medium

Human hepatocellular liver carcinoma (HepG2) cell line and human neuroblastoma cell line, SH-SY5Y, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained at 37 °C in a humidified atmosphere (95% air and 5% carbon dioxide), and they were periodically screened for contamination. HepG2 cells were cultured in Eagle’s Minimum Essential Medium (MEM, Euroclone S.p.A., Pero, MI, Italy), supplemented with 10% Fetal Bovine Serum (FBS, Euroclone S.p.A., Pero, MI, Italy), 1% L-glutamine (Euroclone S.p.A., Pero, MI, Italy), 100 U/mL penicillin/streptomycin (Euroclone S.p.A., Pero, MI, Italy), 1% Non-Essential Amino Acids (NEAA, Euroclone S.p.A., Pero, MI, Italy). SH-SY5Y cells were grown in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM)—high glucose (Euroclone S.p.A., Pero, MI, Italy) and Ham’s F12 Medium (Euroclone S.p.A., Pero, MI, Italy) supplemented with 10% FBS, 1% L-glutamine and 100 U/mL penicillin/streptomycin. For cell assays, cells were trypsinized using Trypsin-EDTA 1X in PBS (Euroclone S.p.A., Pero, MI, Italy).

The human gastric cancer cell line AGS was purchased from the American Type Culture Collection (CRL-1739; Rockville, MD, USA). AGS cells were cultured in HAM’S F12 medium (Euroclone, Via Figino, Italy) supplemented with l-Glutamine 2 mM, 10% heat-inactivated fetal bovine serum (Euroclone), 100 U/mL of penicillin, and 100 µg/mL of streptomycin (Euroclone). The human clone hyper-expressing TFF1 under doxycycline induction (AGS-AC1) was selected from the AGS cell line as previously described [36 (link)]. The AGS-AC1 clone was cultured in DMEM medium (Euroclone) supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin, 100 µg/mL of streptomycin, and 600 µg/mL of neomycin (Euroclone). TFF1 expression was induced with 1 µg/mL of doxycycline. All cell lines were maintained at 37 °C in a 5% CO₂ atmosphere.

Fresh tissue acquired from mastectomies of 8 patients (age 40–89) were collected at the University of Palermo and Fondazione IRCCS INT of Milan, in accordance with ethical standards. Breast tumor cells were purified from fresh tissue via enzymatic digestion as previously described [48 (link)]. Thereafter, single cell suspensions were plated in ultra-low attachment flasks (Corning) at a density of 1×10⁵/ml and grown in a medium supplemented with bFGF (10 ng/ml, Sigma) and EGF (20 ng/ml, Sigma). To induce differentiation, cells were cultured in adherent conditions in Ham's/F-12 medium (Euroclone), supplemented with 5% fetal bovine serum (FBS), insulin (25μg/ml, Sigma) and hydrocortisone (1mg/ml, Sigma). MCF7 cells were purchased from ATCC and cultured in DMEM supplemented with 10% fetal bovine serum (Sigma).