Alanyl glutamine

RPMI 8866 cells (Sigma, St. Louis, MI, USA) were grown at 37 °C in RPMI 1640-Gluta-Max medium (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), sodium pyruvate (PanEco, Singapore), and an antibiotic and antimycotic solution (Gibco, Waltham, MA, USA).
HeLa cells with integrated LTR-TurboRFP (Kurnaeva et al., 2022 (link)) were grown in Dulbecco’s modified Eagle’s medium supplemented with alanyl-glutamine (Paneco, Singapore), 10% fetal calf serum (HyClone, Logan, UT, USA) and an antibiotic and antimycotic solution (Gibco, Waltham, MA, USA). The transactivation assay based on fast-maturing TurboRFP protein was described elsewhere (Kurnaeva et al., 2022 (link)). The expression of EGFP and TurboRFP was analyzed using a FACS Aria SORP instrument (BD Biosciences, San Jose, CA, USA).

Experiments were conducted on the MCF7 human breast cancer cell line (18 (link)) (cat. no. HTB-22^™; American Type Culture Collection), authenticated by morphology and STR profiling through ‘Gordiz’ (http://gordiz.ru/, accessed on February 1, 2022). The cells were cultured at 37˚C with 5% CO₂ in DMEM containing 4.5 g/l glucose (cat. no. СC420-02; PanEco), alanyl-glutamine (cat. no. Ф005; PanEco) and 7% fetal bovine serum (FBS) (cat. no. SV30160.03; HyClone; Cytiva). The response of the cells to tamoxifen (cat. no. 27190; Cayman Chemical Company) was assessed by treating them with tamoxifen for 3 days, followed by evaluating viability using the MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (cat. no. A2231; PanReac AppliChem) (19 (link)) modified as previously described (20 ). Dimethyl sulfoxide (DMSO) (cat. no. 191954; PanReac AppliChem) served as the solvent for the assay. Ultrapure water for the experiments was prepared using a Milli-Q water purification system (Merck KGaA).

The human HEK293T (CRL-3216), HEK293T/17 (CRL-11268), HeLa (CCL-2) obtained from ATCC and mouse DC2.4 (SCC142, Millipore, Burlington, MA, USA) cell lines maintained at 37 °C in a humidified atmosphere with 5% CO₂. HEK293T and HeLa cells were cultured in DMEM (Gibco, Waltham, MA, USA) containing alanyl-glutamine (Paneco, Moscow, Russia) and supplemented with 10% FBS (HyClone, Logan, UT, USA) along with penicillin and streptomycin (Paneco, Moscow, Russia). DC2.4 cells were cultured in RPMI (Gibco, USA) medium with the same supplements. HEK293T/17 cells were cultured in DMEM/F12 (1:1) supplemented with 10% FBS, sodium pyruvate, Glutamax, antibiotic/antimycotic solution, and non-essential amino acids (all reagents from Gibco, USA). To prevent mycoplasma contamination, all cell cultures were constantly tested using the Myco-visor kit (Biolabmix, Novosibirsk, Russia).