Bt 549

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, China

The BT-549 is a laboratory equipment product designed for various scientific applications. It functions as a cell line derived from a human breast carcinoma. The BT-549 cell line is commonly used in research settings.

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122 protocols using bt 549

Cell Line Maintenance and Transfection Protocol

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MDA-MB-231, BT-549, Hs578T, MDA-MB-468, MDA-MB-453, HCC1395, and HEK293 cells were purchased from ATCC (Manassas, VA, USA), SUM159PT were obtained from Dr. Joe Gray (OHSU) and HUVECs were purchased from EMD-Millipore (Billerica, MA, USA). All cell lines were validated by STR profiling and confirmed to be free of mycoplasma contamination. MDA-MB-231, Hs578T, MDA-MB-468, MDA-MB-453, and HEK293 cells were grown in high glucose DMEM (Gibco, Thermo Fisher, Waltham, MA, USA) supplemented with 10% FBS (Atlanta Biologicals, Flowery Branch, GA, USA). BT-549 and HCC1395 cells were grown in RPMI (Gibco) supplemented with 10% FBS. SUM159PT were grown in Hams-F12 media (Gibco) supplemented with 5% FBS, 10mM HEPES, 4μg/mL insulin, and 0.5μg/mL hydrocortisone. HUVECs were grown in complete EndoGRO medium (Millipore).
MiRIDIAN microRNA mimics as well as ON-TARGETplus SMARTpool and ON-TARGETplus individual siRNAs (listed in Supplementary Table 2) were purchased from GE Dharmacon (Lafayette, CO, USA), and transfected using Lipofectamine 2000 or Lipofectamine 3000 (Life Technologies, Carlsbad, CA, USA) according to manufacturer’s protocol at a final concentration of 25nM. BT-549 cells were transfected with LF2000 on two consecutive days to increase transfection efficiency. Phase images of cells were taken on an EVOS-FL scope (Thermo Fisher).

Langer E.M., Kendsersky N.D., Daniel C.J., Kuziel G.M., Pelz C., Murphy K.M., Capecchi M.R, & Sears R.C. (2017). ZEB1-repressed microRNAs inhibit autocrine signaling that promotes vascular mimicry of breast cancer cells. Oncogene, 37(8), 1005-1019.

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Cell Line Authentication and Mycoplasma-Free Culture

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Breast cancer cell lines MDA‐MB‐231 (RRID: CVCL_0062), MCF‐7 (RRID: CVCL_0031) and BT‐549 (RRID: CVCL_1092) were obtained from American Type Culture Collection (ATCC, Manassas, VA). Hair‐follicle‐derived stem cell and dermal fibroblast were isolated as described previously [27 (link)]. All cells except BT‐549 were cultured in Dulbecco's Modified Eagle medium (DMEM) supplemented with 1% (v/v) antibiotic‐antimycotic cocktail (Thermo Fisher Scientific, Grand Island, NY, USA) and 10% (v/v) fetal bovine serum (Atlanta Biologicals, Norcross, GA, USA). BT‐549 was cultured in RPMI (Thermo Fisher Scientific) supplemented with 1% (v/v) antibiotic‐antimycotic cocktail, 10% (v/v) fetal bovine serum and 0.023 U·mL⁻¹ insulin. All cell lines have been authenticated in the past 3 years by STR profiling at ATCC. According to the vendor, the commercially available PowerPlex® 18D kit (Promega, Madison, WI, USA) was used to amplify 17 short tandem repeat (STR) loci plus the gender‐determining locus, Amelogenin. Samples were then processed with the ABI Prism® 3500xl Genetic Analyzer (Thermo Fisher Scientific), and data were analyzed using GeneMapper® ID‐X software (Thermo Fisher Scientific) to confirm cell identity and purity. Finally, all experiments were performed with mycoplasma‐free cells.

Liu Y., Lei P., Samuel R.Z., Kashyap A.M., Groth T., Bshara W., Neelamegham S, & Andreadis S.T. (2023). Cadherin‐11 increases tumor cell proliferation and metastatic potential via Wnt pathway activation. Molecular Oncology, 17(10), 2056-2073.

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Breast Cancer Cell Line Treatment and 4EBP1 Overexpression

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The human breast cancer cell lines MDA-MB-231 and BT549 were purchased from the American Type Culture Collection. Cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.), containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), and 100 µg/ml streptomycin and 100 U/ml penicillin (Gibco; Thermo Fisher Scientific, Inc.) and placed at 37˚C in a humidified incubator containing 5% CO₂. MDA-MB-231 and BT549 cells were treated with arctigenin at various concentrations (5, 10, 20 and 40 µM) or vehicle as the control for 24, 48 and 72 h.
For 4EBP1 overexpression, 50 nM pcDNA3.1-4EBP1 or pcDNA3.1-NC (Invitrogen; Thermo Fisher Scientific, Inc.) were diluted by 250 µl of serum-free Opti-MEM and incubated at room temperature for 5 min and mixed with 5-µl aliquot of Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) for another 20 min at room temperature and added to culture well of MDA-MB-231 or BT549 cell (5x10⁴/well). Subsequently, the cells were cultured for 6-8 h at 37˚C with 5% CO₂, the complete medium was refreshed and the cells were cultured for a further 48 h prior to the following experiments.

Luo W., Wang F., Luo H, & Liu H. (2021). Arctigenin inhibits human breast cancer cell proliferation, migratory and invasive abilities and epithelial to mesenchymal transition by targeting 4EBP1. Experimental and Therapeutic Medicine, 21(6), 547.

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Breast Cancer Cell Line Cultivation

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Breast cancer cell lines were kindly provided by the Center for Cancer Immune Therapy, Herlev Hospital, Denmark (BT-549 and MDA-MB-231) or purchased from ATCC (HCC1937, Cat#CRL-2336) and DSMZ (EFM192A, Cat#ACC258) and grown in R10 (RPMI + GlutaMAX^TM, Gibco, Cat#61870010 + 10% FCS) including 1% penicillin-streptomycin (P/S, Sigma-Aldrich, Cat#P0781).

Viborg N., Ramskov S., Andersen R.S., Sturm T., Fugmann T., Bentzen A.K., Rafa V.M., Straten P.T., Svane I.M., Met Ö, & Hadrup S.R. (2019). T cell recognition of novel shared breast cancer antigens is frequently observed in peripheral blood of breast cancer patients. Oncoimmunology, 8(12), e1663107.

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Cultivation Conditions for Triple-Negative Breast Cancer Cell Lines

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The TNBC cell lines BT549, MB468, MB231, HCC1937, and 4T1 were purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China). HEK-293T cell line was purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China). SUM149 and SUM159 cells were obtained from the State Key Laboratory of Biotherapy of Sichuan University (Chengdu, China). MB468, MB231, and SUM149 cells were cultured in a humidified of 5% CO2 at 37°C in DMEM (Gibco) medium supplementing with 10% fetal bovine serum (FBS) (NATOCOR) and 1% penicillin-streptomycin (100 μg/mL, Hyclone). BT549, HCC1937, SUM159, and 4T1 cells were cultured in a humidified of 5% CO2 at 37°C in RPMI (Gibco) medium supplementing with 10% fetal bovine serum and 1% penicillin-streptomycin. All cell lines were confirmed mycoplasma negative by end-point PCR.

Wang X., Yu J., Liu X., Luo D., Li Y., Song L., Jiang X., Yin X., Wang Y., Chai L., Luo T., Jing J, & Shi H. (2022). PSMG2-controlled proteasome-autophagy balance mediates the tolerance for MEK-targeted therapy in triple-negative breast cancer. Cell Reports Medicine, 3(9), 100741.

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Establishing Tamoxifen-Resistant Breast Cancer Cell Lines

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Human breast cancer cell line MCF-7 (tamoxifen-sensitive), T47D (tamoxifen-sensitive), BT-549, SK-BR-3, MDA-MB-231 and MCF-10A were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The identities of cell lines were confirmed by using DNA profiling (short tandem repeat, STR). MCF-7/TAM-1 and MCF-7/TAM-2 (tamoxifen-resistant) cell lines were established from MCF-7 cells after the following continuous exposure to 1 μM of TAM (Sigma, St. Louis, MO, USA) for more than 1 year. MCF-10A were cultured in DMEM/F12 (1:1) supplemented with 5% heat-inactivated equine serum, l0 μg/ml insulin 20 ng/ml EGF, 100 ng/ml cholera toxin, 0.5 μg/ml hydrocortisone and 2 mmol/l l-glutamine. BT-549 cells were cultured as previously described.^{42 (link)} All other cell lines were maintained in DMEM medium supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) at 37 °C in a 5% CO₂ humidified incubator. Cells were grown in monolayer and passaged routinely 2–3 times a week. Dimethyl sulfoxide (DMSO) and EST was purchased from Sigma.

Zhu J., Zou Z., Nie P., Kou X., Wu B., Wang S., Song Z, & He J. (2016). Downregulation of microRNA-27b-3p enhances tamoxifen resistance in breast cancer by increasing NR5A2 and CREB1 expression. Cell Death & Disease, 7(11), e2454-.

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Culture of Breast Cancer Cell Lines

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Human breast cancer cell lines MDA-MB-231 and BT549 were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). MDA-MB-231 cells were cultured in Leibovitz’s L-15 medium supplemented with 10% fetal bovine serum (Invitrogen, Shanghai, China); BT549 cells were cultured in RPMI-1640 (GIBCO, Shanghai, China) supplemented with 10% fetal calf serum. Cells were in a 37 °C humidified incubator with 95% air, 5% CO₂.

Zhang Y.C., Huo F.C., Wei L.L., Gong C.C., Pan Y.J., Mou J, & Pei D.S. (2017). PAK5-mediated phosphorylation and nuclear translocation of NF-κB-p65 promotes breast cancer cell proliferation in vitro and in vivo. Journal of Experimental & Clinical Cancer Research : CR, 36, 146.

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Culturing Human Breast Cancer Cell Lines

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Human breast cancer cell lines, MDA-MB-231 and BT-549, were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and were maintained according to the ATCC's recommendation. MDA-MB-231 cells were maintained in L-15 medium; BT-549 cells were routinely maintained in RPMI-1640 medium (Life Technologies/Gibco, Grand Island, NY, USA). Both media were supplemented with 10% FBS (Life Technologies/Gibco), 100 U/mL penicillin and 100 mg/mL streptomycin (Life Technologies/Gibco, Gaithersburg, MD, USA). BT-549 cells were incubated at 37°C in 5% CO₂; MDA-MB-231 cells were grown in the absence of CO₂. GLA (99.0% purity) was purchased from Sigma Chemical (St. Louis, MO, USA).

Ye X., Jiang F., Li Y., Mu J., Si L., Wang X., Ning S, & Li Z. (2014). Retracted: Glabridin attenuates the migratory and invasive capacity of breast cancer cells by activating microRNA-200c. Cancer Science, 105(7), 875-882.

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Breast Cancer Cell Line Culture Protocols

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The breast cancer cell lines 4T1, MDA-MB-231, BT549 and HEK 293 human kidney cells were obtained from the American Type Culture Collection (ATCC^®). 4T1, MDA-MB-231 and HEK 293 cells were cultured in high glucose DMEM (Gibco^®) supplemented with 10% FBS (Gibco^®). BT549 cells were cultured in RPMI-1640 (Gibco^®) supplemented with 10% FBS. All cells were maintained at 37°C in a humidified 5% CO₂ atmosphere. The ALK5 inhibitor A8301, HDAC8 inhibitor PCI-34051 and paclitaxel were obtained from MedChemExpress (MCE^®). Other HDAC inhibitors mentioned in manuscript were from Selleck.

Tang X., Li G., Su F., Cai Y., Shi L., Meng Y., Liu Z., Sun J., Wang M., Qian M., Wang Z., Xu X., Cheng Y.X., Zhu W.G, & Liu B. (2020). HDAC8 cooperates with SMAD3/4 complex to suppress SIRT7 and promote cell survival and migration. Nucleic Acids Research, 48(6), 2912-2923.

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Cytotoxicity Assay of Breast Cancer Cell Lines

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The human breast cell lines BT549, Hs578T, MDA-MB-231, and MDA-MB-468 were obtained from American Type Culture Collection (Rockville, MD, USA). Cells were cultured in RPMI 1640 medium (Mediatech Inc., Manassas, VA, USA) for BT549 and 4T1 cells, in 1:1 DMEM/F12 (Gibco, Thermo Scientific, Waltham, MA, USA) for MDA-MB-468 cells, and in DMEM medium (Mediatech Inc., Manassas, VA, USA) for MDA-MB-231 and Hs578 cells. Culture media were supplemented with 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA, USA) and 1% penicillin/streptomycin (Gibco, Thermo Scientific, Waltham, MA, USA).
Cells (4000 per well) were plated in a 96-well plate format in 100 μL growth medium. Cells were treated with dimethyl sulfoxide (DMSO) or drugs the next day at the indicated concentrations and incubated for an additional 3 days. Viable cells were determined by the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Promega, Madison, WI, USA) [33 (link)]. After treatment, the media were removed and MTT dye was added to each well and incubated for 4 h according to the manuscripter’s instruction. The resulting formazan crystals were dissolved in DMSO after removal of the media. Absorbance was read at 570 nm. The half maximal inhibitory concentration IC₅₀ was determined using the Calcusyn software package (Biosoft, Ferguson, MO, USA).

Wen W., Marcinkowski E., Luyimbazi D., Luu T., Xing Q., Yan J., Wang Y., Wu J., Guo Y., Tully D., Han E.S., Yost S.E., Yuan Y, & Yim J.H. (2019). Eribulin Synergistically Increases Anti-Tumor Activity of an mTOR Inhibitor by Inhibiting pAKT/pS6K/pS6 in Triple Negative Breast Cancer. Cells, 8(9), 1010.

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