Hbl 100

Human breast cancer cell lines MCF-7, MDA-MB-231, BT-549, SK-BR-3, MDA-MB-468 and T-47D and normal breast cell line HBL-100 were purchased from Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai Institute of Cell Biology, Shanghai, China). The cell lines MCF-7, MDA-MB-231, SK-BR-3, T-47D and HBL-100 were cultured in Dulbecco's modified Eagle medium (DMEM, Gibco, Thermo Fisher Scientific, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, MA, USA). BT-549 and MDA-MB-468 were cultured in Roswell Park Memorial Institute 1640 medium (RPMI164, Gibco, Thermo Fisher Scientific, MA, USA) supplemented with 10% FBS. All cell lines were cultured in a humidified incubator containing 5% CO₂ at 37 °C.

Human mammary gland epithelial adenocarcinoma cell lines (luminal subtype A: BT474 (ATCC HTB-20), BT483 (ATCC HTB-121), MCF-7 (ATCC HTB-22), T47D (ATCC HTB-133), and ZR75-1 (ATCC CRL-1500) [42 (link)]; luminal subtype B: AU565 (ATCC CRL-2351), MDA-MB-453 (ATCC HTB-131), and SKBR3 (ATCC HTB-30) [43 (link)–45 (link)]; basal subtype: MDA-MB-231 (ATCC HTB-26) HS578T (ATCC HTB-126) [46 (link)]); the normal human breast epithelial cell line MCF-10A (ATCC CRL-10317); and an immortalized cell line obtained from a primary culture of cells derived from an early lactation sample of human milk, HBL100 (ATCC HTB-124), were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). MCF-10A cells were maintained in MCF-10A culture medium consisting of DMEM/F12 (Thermo Fisher Scientific, Passau, Germany) supplemented with 20 ng/mL epidermal growth factor, 10 g/mL insulin, 0.5 g/mL hydrocortisone, and 1X non-essential amino acids (Thermo Fisher Scientific). BT474, BT483, MCF-7, MDA-MB-453, MDA-MB-231, and HBL-100 cells were maintained in DMEM (Thermo Fisher Scientific). T47D, ZR-75 AU-565, SKBR3, and HS-578T cells were maintained in DMEM/F12 (Thermo Fisher Scientific). The cells were cultured according to standard protocols [21 (link)].

The human breast cancer cell lines MCF-7 and MDA-MB-231, human mammary epithelial cells with integrated SV40 gene (HBL-100) as well as non-malignant mammary epithelial cell line MCF-10A were obtained from ATCC (Manassas, VA, United States). All the above cell lines have been identified by short tandem repeat analysis. RPMI-1640 medium (Gibco Life Technologies, Lofer, Austria) for MCF-7 cells and Dulbecco's Modified Eagle medium (DMEM, Gibco Life Technologies, Lofer, Austria) for MDA-MB-231 cells and HBL-100 cells were applied in exception to 10% fetal bovine serum (Gibco) and 1% penicillin and streptomycin (Gibco). And MCF-10A cells were maintained in DMEM/F12 medium (Gibco) supplemented with 5% horse serum (HyClone, Logan, Utah, United States), 1% penicillin and streptomycin, 20 ng/ml recombinant human epidermal growth factor (BD Bioscience, Bedford, MA, United States), 0.5 μg/ml hydrocortisone (STEMCELL Technologies, Vancouver, Canada), 100 ng/ml cholera toxin (MACGENE, Beijing, China) and 10 μg/ml insulin (Sigma, St. Louis, MO, United States). DMEM/F12 medium for the CSCs derived from MDA-MB-231 and MCF-7 cells were employed in addition to B27 (Invitrogen, Carlsbad, CA, United States), 5 μg/ml of insulin, 20 ng/ml of hEGF, 1% penicillin and streptomycin and 0.4% BSA (Sigma) at 37°C with 5% CO₂.

The human normal breast epithelial cell line (HBL-100), 5 TNBC cell lines (MDA-MB-231, BCap37, Hs 578 T, BT-549, HCC1937) and the non-TNBC cell line (MCF-7) were purchased from the Cell Bank of the Chinese Scientific Academy. HBL-100, BCap37, BT-549, HCC1937 and MCF-7 were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, 31800105, Life technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Biological Industries, 04-0101-1, Cromwell, CT, USA). MDA-MB-231 were cultured in Leibovitz’s L-15 medium (Gibco, 11415114) with 10% FBS. Hs 578 T were cultured in Dulbecco’s Modified Eagle’s (DMEM) Medium (ATCC® 30-2002™), with 10% FBS. All cells were incubated at 37 °C with 5% CO₂ in a water-jacketed incubator (Thermo Scientific, Waltham, MA, USA). The cell culture medium was changed every two days, and experiments were initiated when cells showed logarithmic growth at 70–80% confluence.

The Chinese Cell Repository provided MDA-MB-231, BT-549, SUM1315MO2, and ZR-75–1 breast cancer cell lines as well as human breast epithelial cell lines (HBL-100) (Shanghai, China).
All cells were grown at 37°C with 5% CO₂ in DMEM (Gibco) with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin solution (Gibco). These cells were transfected using the Lipofectamine3000 (Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer’s procedure with previously generated short interfering RNAs (Hippobiotec, Huzhou, China) targeting gene GTPBP4. Supplementary Table S1 lists the siRNA sequences for the gene GTPBP4. All data were presented as the means ± SD of three independent experiments.