Dmem f12 medium

The cell lines FHC (ATCC® CRL-1831), DLD1 (ATCC® CCL-221) and HEK293T (ATCC® CRL-3216) were obtained from the American Tissue Culture Collection (ATCC). FHC (CRL-1831) cells were cultured in DMEM/F12 medium (Gibco, Invitrogen) supplemented with 25 mM HEPES, 10 ng/ml cholera toxin (Sigma Aldrich, Germany), 0,005 mg/ml insulin, 0,005 mg/ml transferrin, 100 ng/ml hydrocortisone (all from Sigma Aldrich, Germany), and 10% FBS (Gibco, Invitrogen). DLD1 colon carcinoma cells were cultured in RPMI 1640 medium (Gibco, Invitrogen) supplemented with 10% FBS (Gibco, Invitrogen), 2 mM glutamine and 1% Pen-Strep. HEK293T packaging cells were cultivated in DMEM medium supplemented with 10% FBS (Gibco, Invitrogen), 2 mM glutamine and 1% Pen-Strep. Reprogrammed cell lines iPSC-CRL-1831, derived from human colon normal cell line CRL-1831, and CSC-DLD1, from human colon carcinoma cell line DLD1, respectively, were cultured in stem cell medium (SCM) in hESC qualified Matrigel (BD Biosciences) modified plates. SCM consisted of DMEM/F12 medium (Gibco, Invitrogen) supplemented with 1× MEM-NEAA, 1× Glutamax, 50 μM β-Mercaptoethanol, 10 ng/μl bFGF (all from ThermoFisher Scientific), and 20% knockout serum replacement (ThermoFisher Scientific) SCM medium was changed daily, and cells were passaged by manual dissociation with EDTA or dispase.

For primary mGC isolation, ovaries were removed from 21-day-old immature ICR mice and were punctured with 25-gauge needles. The cells were pooled and filtered with a 40-μm cell strainer to remove oocytes. The isolated cells were determined to have 95% purity via follicle-stimulating hormone receptor staining as described previously.^{22 (link)} The mGCs were cultured in DMEM/F12 medium (Gibco BRL/Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (HyClone, South Logan, UT, USA), 1 mM sodium pyruvate, 2 mM glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin. KGN cells, a human ovarian GC-like tumor cell line, were maintained in DMEM/F12 medium (Gibco BRL/Invitrogen) supplemented with 10% newborn calf serum (Gibco BRL/Invitrogen), 100 IU/ml penicillin, and 100 μg/ml streptomycin. All cells were maintained at 37 °C in a humidified environment with 5% CO₂. In some experiments, GCs were cultured in the presence of H₂O₂ (Sigma, St. Louis, MO, USA), nicotinamide (NAM; Sigma), trichostatin A (TSA; Sigma), or SIRT1 activator 3 (SA3; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Wound healing assays were performed as previously described.^{8 (link)} Invasion analyses were performed using Boyden chambers (Millicell) with 8 µm pore-sized membranes, coated with Matrigel (BD Bioscience). Control or GW8510 treated H295R cells (2.5 × 10⁵ cells) were seeded in the upper chamber in minimum medium DMEM-F12 medium (Invitrogen) containing penicillin/streptomycin (100 mg ml⁻¹) and l-glutamine (2 mM). The lower chamber contained DMEM-F12 medium (Invitrogen) supplemented with penicillin/streptomycin (100 mg ml⁻¹), l-glutamine (2 mM), 10% FBS (Biowest) and 5% SR3 (S2640, Sigma). Cells were incubated for 48 h before fixation using Paraformaldehyde 4% and staining with Haematoxylin. Clonogenic growth assays were performed as described in Drelon et al.^{8 (link)}

BC cells were cultured in six-well plates in the glutamine-free DMEM/F-12 medium (Invitrogen; Thermo Fisher Scientific, USA). Following collection and counting, the cells were incubated with 200 nM [³H]-L-glutamine (PerkinElmer; Thermo Fisher Scientific, USA) in glutamine-free DMEM/F-12 medium (Gibco; Thermo Fisher Scientific, USA) for 15 min at 37˚C in the presence of curcumin or with curcumin + SLC1A5 small interfering RNA (siRNA). The cells were collected, transferred to filter paper using a 96-well plate harvester, dried, and exposed to scintillation fluid. The counts were measured using a liquid scintillation counter (PerkinElmer; Thermo Fisher Scientific).

Human EnSCs (#T0533, Applied Biological Materials Inc)^{23 (link),64 (link),65 (link)} were cultured at 37 °C in a humidified 5% CO₂ atmosphere in DMEM/F-12 medium (#11039-021, Invitrogen) containing 10% (v/v) dextran-coated charcoal striped (#C6241, Sigma) fetal bovine serum (#10270-106, Invitrogen). Human BeWo cells (#86082803, Sigma)^{66 (link),67 (link)}, a human trophoblast cell line, were cultured in DMEM/F-12 medium (#11039-021, Invitrogen) containing 10% (v/v) fetal bovine serum (#10270-106, Invitrogen).

mESCs were cultured on feeder cells or gelatin in the 2i (CHIR99021, PD0325901)/LIF medium and were differentiated in the N2B27 medium. 2i/LIF medium: 500 mL of Neurobasal medium (Gibco, Shanghai, China, 21103049), 500 mL of DMEM/F12 medium (Gibco, 11330057) that was supplemented with 10 mL N2 (Thermo Fisher Scientific, 17502048), 20 mL B27 (Thermo Fisher Scientific, 12587010), 1 μM PD0325901 (Selleck, S1036), 3 μM CHIR99021 (Selleck, S1263), 0.1 mM β-mercaptoethanol (Gibco, 21986), 1% penicillin/streptomycin (Gibco, 15104122), and 10⁶ unit/L LIF (Millipore, ESG1106). N2B27 medium: 500 mL of Neurobasal medium (Gibco, 21103049), 500 mL of DMEM/F12 medium (Gibco, 11330057) supplemented with 10 mL N2 (Thermo Fisher Scientific, 17502048), 20 mL B27 (Thermo Fisher Scientific, Shanghai, China, 12587010), 0.1 mM β-mercaptoethanol (Gibco, 21986), and 1% penicillin/streptomycin (Gibco, 15104122).
The feeder cells were cultured in DMEM (Gibco, 11960) that was supplemented with 10% fetal bovine serum (Gibco, 10099), 1 mM sodium pyruvate (Gibco, 11360070), 1% nonessential amino acids (Gibco, 11140050), and 1% penicillin/streptomycin (Gibco, 15104122). All the cell lines were incubated at 37 °C in a humidified atmosphere containing 5% CO₂ and were negative for mycoplasma contamination.