Zr 75 30

HEK293T, MDA-MB-231, MCF7, ZR-75-30, MCF10A, Hela, U2OS, A549, and 4T1 cells were obtained from ATCC (Manassas, USA). Details were described in the supplemental materials and methods.

The murine breast cancer cell line Met1 was isolated from MMTV-PyMT mice, while Eo771 (CRL-3461) and 4T1 (CRL-2539) breast cancer cell lines were acquired from the American Type Culture Collection (ATCC). Human breast cancer cell lines ZR75-30 (CRL-1504), BT-474 (HTB-20), SK-BR-3 (HTB-30), MCF7 (HTB-22), MDA-MB-231 (CRM-HTB-26), BT-20 (HTB-19), and T47-D (HTB-133) were also acquired from ATCC. BT-20 cell line is under the list of known misidentified cell lines provided from the International Cell Line Authentication Committee (ICLAC). However, BT-20 cell was used as one of the triple negative breast cancer cells, which displayed limited responses to high glucose, and thus, it does not compromise our results. Cell culture medium was supplemented with 10% FBS and 1% penicillin/streptomycin, and cells were maintained in a 5% CO₂ humidified incubator. To establish a cell line-based model system for studying hyperglycemia-driven breast cancer cells, a low glucose (1 g/L) adaptation period was applied. All the breast cancer cells were maintained in low glucose DMEM for at least 3 days and then transferred to high glucose (4.5 g/L) DMEM until the indicated time points.

The breast cancer cell lines: MDA-MB-231, MCF-7, BT-549, ZR-75-30, SKBR-3, and T47D (ATCC, Manassas, VA, USA) were maintained as previously described (Zhang et al., 2019 (link)). The normal mammary epithelial cell line MCF-10A was also maintained as previously described (Debnath, Muthuswamy & Brugge, 2003 (link)). All cell lines were cultured in a humidified incubator at 37 °C with an atmosphere containing 5% CO₂.

The cell lines used in this study were purchased from the American Type Culture Collection (ATCC, Manassas, VA), including the human embryonic kidney cell line HEK-293 (ATCC® CRL-1573™), human breast epithelial cell line ZR-75-30 (ATCC® CRL-1504™), human AML cell line THP-1 (ATCC TIB-202), human lymphoblast line MV4-11 (ATCC® CRL-9591™), and human cutaneous T-lymphocyte line H9 (ATCC® HTB-176™). The HEK-293 cell line was cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco-BRL, Grand Island, NY, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin. MV4-11 cells were cultured in Iscove’s modified Dulbecco’s medium (Invitrogen) supplemented with 10% heat-inactivated FBS. Other cell lines were cultured in Roswell Park Memorial Institute 1640 medium (Invitrogen) supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin. All cell lines were authenticated by short tandem repeat profiling <6 months ago, which is when this project was initiated. All cell lines were passaged for 2 months, to ensure fidelity of the cell line identity. All cells were tested for mycoplasma contamination and all of the cell lines in this study were confirmed to have received treatment for mycoplasma contamination.

MCF10A, MDA-MB-231, MDA-MB-453, MCF-7, and ZR-75-30, Cells were purchased from ATCC (Manassas, USA). The cells were cultured in DMEM medium supplemented with 10% FBS and incubated under suitable conditions (37°C; 5% CO2).