L glutamine

HaCaT cells obtained from the Cell Lines Service (Cell Lines Service, Eppelheim, Germany), were maintained at 37 °C under 5% CO₂ in complete DMEM containing Dulbecco’s modified Eagle’s medium (DMEM), 10% heat-inactivated fetal bovine serum (FBS), 2 mM of L-glutamine, 100 U/mL of penicillin, and 100 U/mL of streptomycin. DMEM medium, FBS, L-glutamine, penicillin/streptomycin, phosphate-buffered saline (PBS), and trypsin-EDTA were purchased from GIBCO (Gibco, Grand Island, NY, USA).

The FRTL-5 (RRID:CVCL_0265) was obtained from CLS (Cell Lines Service GmbH, Eppelheim, Germany). It was cultured in Ham’s F12 Coon’s modification cell growth medium supplemented with TSH (final concentration 16.5 ng/mL), L-glutamine, and FBS (Cell Lines Service GmbH, Eppelheim, Germany).
The BCPAP (human PTC cell line, RRID:CVCL_0153) was obtained from the German Collection of Microorganisms and Cell Cultures (Leibniz Institute DSMZ, Braunschweig, Germany); the K1 (human PTC cell line, RRID:CVCL_2537) was obtained from the European Collection of Authenticated Cell Cultures (ECACC, Sigma-Aldrich S.r.l., Milan, Italy).
The BCPAP and K1 cell lines were cultured in RPMI 1640 (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum (FBS) (Gibco), L-glutamine (2 mM), and penicillin/streptomycin (100 IU/mL and 100 μg/mL, respectively).
Adherent monolayer cultures were maintained in T75 culture flasks and incubated at 37 °C with 5% CO₂ until they reached 85% confluency. Cells were detached using 0.025% trypsin (Sigma-Aldrich) and plated into T75 flasks at a density of 2 × 10⁶ cells.

We obtained the SK-BR3 cell line and the BT474 cell line from Cell Line Service (Eppelheim, Germany). The SK-BR3 cells were cultured in Dulbecco's MEM (Biochrom) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin at 37 °C and 5% CO₂ in a humidified chamber. BT474 cells were cultured in DMEM:Hams F12 (1:1) supplemented with L-glutamine, Insulin and FBS (Cell Lines Service, Eppelheim, Germany). The medium was renewed every 2–3 days. They were cultured until 70–80% confluence. Cell harvesting was performed with Accutase treatment for 10 min in a humidified chamber at 37 °C and 5% CO₂. Subsequently, the cells were rinsed with sterile phosphate-buffered saline (PBS) and counted using a Neubauer counting chamber. The cells were seeded at a density of 30,000–40,000 per well in culture slides (Falcon CultureSlides, Corning Life Science, New York, USA, Cat.# 354108). Mycoplasm infection was excluded with 4′,6-diamidino-2-phenylindole (DAPI) staining for each test.