Glutamine

Breast cancer cell lines ZR-75-1, BT-474, CAMA-1, SK-BR-3, T-47D, MCF7, MDA-MB-468, BT-549, Hs 578T, MDA-MB-453 and MDA-MB-231 were obtained from the American Type Cell Culture Collection (ATCC). The TSE breast cancer cell line was kindly supplied by Dr Steven Abcouwer (University of Michigan). All breast cancer cell lines were maintained at 37 °C, 5% CO₂ atmosphere, in RPMI 1640 medium containing 2 mM glutamine (Gibco) and supplemented with 10% FBS (Gibco). For glutamine-withdrawal experiments, glutamine-free RPMI 1640 medium (Gibco) supplemented with 10% dialysed FBS (Gibco) was used. Cell lines were periodically tested for Mycoplasma contamination.

Human lung cancer A549 (American Type Culture Collection [ATCC] catalog no. CCL-185, Research Resource Identifier [RRID]: CVCL_0023), human TNBC MDA-MB-436 (ATCC catalog no. HTB-130, RRID: CVCL_0623), and human melanoma A-375 (ATCC catalog no. CRL-1619, RRID: CVCL_0132) cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS; Gibco), 1% glutamine (Gibco), 100 μg/mL streptomycin, and 100 U/mL penicillin (Life Technologies). Murine TNBC cell line 4T1 (ATCC catalog no. CRL-2539, RRID: CVCL_0125) was cultured in RPMI high glucose with 10% FBS (Gibco), 1% glutamine (Gibco), 100 μg/mL streptomycin, and 100 U/mL penicillin (Life Technologies). Prof. Richard Vile kindly provided B16-OVA, a mouse melanoma cell line expressing chicken OVA (Mayo Clinic). B16-OVA cells were cultured in RPMI with 10% FBS (Gibco), 1% glutamine (Gibco), 100 μg/mL streptomycin, and 100 U/mL penicillin (Life Technologies), and 5 mg/mL geneticin (Life Technologies). The cells were cultivated at 37°C, 5% CO₂ in a humidified atmosphere, and regularly tested for mycoplasma contamination.

For glutamine deprivation assay, SKBR3FLAG-KISS1R cells and controls were seeded in 6 cm dishes (400,000 cells each) in glutamine-free RPMI media with dialyzed FBS. Cells were treated with 0.02 mM glutamine, 0.2 mM glutamine, or 2 mM glutamine (Gibco) over 72 h; media was changed every 24 h and cells trypsinized and counted using a hemocytometer at 24 h intervals. For BPTES or CB-839 (Sigma Aldrich) treatment, SKBR3FLAG-KISS1R cells (400,000 cells) were plated in 6 cm dishes. On the following day, these cells were treated with different concentrations of BPTES or CB-839 and cell number counted at 24 h intervals. To determine the effect of c-Myc knockdown on cell growth, SKBR3FLAG-KISS1R cells expressing c-Myc siRNA were cultured in media without glutamine. Media was changed daily and each day cells were counted for each experimental condition.

LNCaP, C4‐2, CWR22res, VCaP, DU‐145, PC‐3 and PC‐3met were cultured in RPMI (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) medium supplemented with 10% foetal bovine serum (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 2 mM glutamine (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). 22rv1 and LNCaP AI cells were maintained in phenol‐free RPMI (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% charcoal‐stripped serum (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 2 mM glutamine. MCF‐7 were maintained in DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% foetal bovine serum (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 2 mM glutamine. CWR22Res cells (hormone‐responsive variant of CWR22 cells) were obtained from Case Western Reserve University, Cleveland, Ohio. All other cell lines were obtained from ATCC. All cells were maintained at 37°C under 5% CO₂ and routinely harvested with trypsin. All cell lines were authenticated by STR DNA profiling and were tested negative for mycoplasma using the Mycoalert mycoplasma detection kit (Lonza, Basel, Switzerland). Cells were kept in culture for a maximum of 10 passages after recovery from frozen vials.

Human embryonic kidney cells 293 (HEK) were cultured and maintained in Dulbecco’s modified Eagle’s medium high glucose (EuroClone, Pero, Italy) containing 10% fetal bovine serum (Gibco-Thermo Fisher Scientific, Waltham, MA, USA), 1% glutamine (Gibco-Thermo Fisher Scientific, Waltham, MA, USA), and 1% penicillin/streptomycin (Gibco-Thermo Fisher Scientific, Waltham, MA, USA), in a humidified atmosphere containing 5% CO₂ at 37 °C.
Glioblastoma U87 cells (U87) were cultured and maintained in Dulbecco’s modified Eagle’s medium low glucose (EuroClone, Pero, Italy) containing 10% fetal bovine serum (Gibco-Thermo Fisher Scientific, Waltham, MA, USA), 1% glutamine (Gibco-Thermo Fisher Scientific, Waltham, MA, USA), and 1% penicillin/streptomycin (Gibco-Thermo Fisher Scientific, Waltham, MA, USA), in a humidified atmosphere containing 5% CO₂ at 37 °C.
Human monocytic cells deriving from an acute monocytic leukemia patient (THP-1) were cultured and maintained in RPMI 1640 medium (EuroClone, Pero, Italy) containing 10% fetal bovine serum (Gibco-Thermo Fisher Scientific, Waltham, MA, USA), 1% glutamine (Gibco-Thermo Fisher Scientific, Waltham, MA, USA), and 1% penicillin/streptomycin (Gibco-Thermo Fisher Scientific, Waltham, MA, USA), in a humidified atmosphere containing 5% CO₂ at 37 °C.

CHO-K1 cells were cultivated in CD CHO medium (Gibco) supplemented with 8 mM glutamine (Gibco), 1x PenStrep (Gibco), and 2 ml/l anticlumping agent (Gibco) at 37ºC, 5% CO₂. CHO cells were harvested during exponential growth at approximately 48 h after seeding with a cell density of 0.2 × 10⁶ cells/ml. HEK cells were cultivated in DMEM medium (Gibco) supplemented with 10% FCS (Thermo Scientific), 4 mM glutamine (Gibco), and 1 x PenStrep (Gibco). For HeLa cells RPMI (Gibco) medium supplemented with 10% FCS, 4 mM glutamine (Gibco) and 1 x PenStrep (Gibco) was used. For northern blot analysis, the following stress conditions were applied: (I) stationary growth phase: for CHO-K1 cells 7 days after seeding with a cell density of 0.2 × 10⁶ cells/ml, for HeLa and HEK cells growth to the overconfluent stage, (II) heat shock treatment: 42°C for 30 min, (III) oxidative stress: incubation with 2 mM H₂O₂ for 2 h, (IV) recovery from oxidative stress: 2 mM H₂O₂ for 2 h followed by 2 h recovery in fresh media, (V) nutritional deprivation by incubation the cells in PBS for 2 h.