Human monocytic cell line THP-1, human breast carcinoma cell lines MDA-MB-231, MDA-MB-468, Hs578T, MCF-7, T47D, and ZR75-1 were purchased from ATCC and Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). Peripheral blood mononuclear cells (PBMCs) were isolated using Histopaque-1077 (10771, Sigma-Aldrich, Castle Hill, Australia), from healthy donors who had agreed and signed an informed consent form approved by the Institutional Review Board of Kaohsiung Medical University Hospital (Kaohsiung, Taiwan). MDA-MB-231, luciferase-expressing MDA-MB-231 (Luc- MDA-MB-231), MDA-MB-468, Hs578T, and MCF-7 cells were cultured in DMEM medium (11875093, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Biological Industries, Cromwell, CT, USA) and 1% Penicillin-Streptomycin-Amphotericin B Solution (03-033-1B, Biological Industries). THP-1, PBMCs, T47D, and ZR75-1 were cultured in RPMI 1640 medium (12100046, Thermo Fisher Scientific) supplemented with 10% FBS and 1% Penicillin-Streptomycin-Amphotericin B Solution. All cells were grown in a 37 °C incubator under 5% CO2 atmosphere at constant humidity.
Mda mb 231
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Germany, United Kingdom, Austria, Australia, Spain, Italy, Canada, France
MDA-MB-231 is a human breast adenocarcinoma cell line derived from a metastatic site. It is a commonly used model for studying breast cancer research.
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Lab products found in correlation
Mda mb 231, by American Type Culture Collection (461 mentions)
Mcf 7, by Thermo Fisher Scientific (405 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (394 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (283 mentions)
Mcf 7, by American Type Culture Collection (264 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (168 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (153 mentions)
Streptomycin, by Thermo Fisher Scientific (136 mentions)
Mda mb 468, by Thermo Fisher Scientific (135 mentions)
Penicillin, by Thermo Fisher Scientific (133 mentions)
Mcf 10a, by American Type Culture Collection (124 mentions)
Rpmi 1640, by Thermo Fisher Scientific (116 mentions)
Sk br 3, by Thermo Fisher Scientific (96 mentions)
Mda mb 468, by American Type Culture Collection (94 mentions)
Bt 549, by Thermo Fisher Scientific (89 mentions)
Mcf 10a, by Thermo Fisher Scientific (83 mentions)
Skbr3, by American Type Culture Collection (72 mentions)
Dmem f12, by Thermo Fisher Scientific (66 mentions)
Mda mb 453, by Thermo Fisher Scientific (61 mentions)
Bt549, by American Type Culture Collection (58 mentions)
843 protocols using mda mb 231
1
Culturing Human Cell Lines for Research
2
Cell Culture Protocols for Breast Cancer and T-Cell Lines
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The human breast cancer cell line MDA-MB-231 used in this study was obtained from the American Type Culture Collection (ATCC). MDA-MB-231 and the 2 constructed cell lines (MDA-MB-231 PD-L1 OE and MDA-MB-231 PD-L1 KO) were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and 100 U/mL penicillin-streptomycin. The Jurkat cell line obtained from ATCC was maintained in ATCC-formulated RPMI-1640 supplemented with 10% FBS (Gibco) and 100 U/mL penicillin-streptomycin. All cell lines were cultured at 37°C in a humid atmosphere supplemented with 5% CO2.
3
Cancer Cell Line Cultivation Protocol
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Two human-prostate-carcinoma-derived cancer cell lines (PC-3 and LNCaP) were cultivated in Roswell Park MEMorial Institute (RPMI) 1640 culture medium (ThermoFisher Scientific, Waltham, MA, USA), and two human-breast-adenocarcinoma-derived cancer cell lines (MCF-7 and MDA-MB-231) and normal human dermal fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM, ThermoFisher Scientific). All culture media were supplemented with 10% (v/v) fetal bovine serum (FBS, ThermoFisher Scientific) and a mix solution of 100 U/mL penicillin and 100 µg/mL streptomycin (ThermoFisher Scientific). Additionally, the MCF-7 and MDA-MB-231 culture media were also supplemented with 1% (v/v) MEM, a non-essential amino acid (ThermoFisher Scientific). The cell cultures were maintained at 37 °C in a humidified atmosphere of 5% (v/v) CO2. All cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cell culture characteristics are summarized in Table 1 . For more details, please consult Figure S1 .
4
Breast Cell Line Manipulation Protocol
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Human normal breast cell line MCF-10A and breast cancer cell lines HCC1937, MDA-MB-231 and MCF-7 were obtained from BeNa Culture Collection (Beijing, China). MCF-10A and HCC1937 cells were cultured in the completely culture medium containing 90% RPMI-1640 (Thermo Fisher Scientific, Waltham, MA, USA) and 10% FBS (Thermo Fisher Scientific); MDA-MB-231 cells were cultured in 90% L-15 cell culture medium (Thermo Fisher Scientific) and 10% FBS; MDA-MB-231 cells and MCF-7 cells were cultured in minimum essential medium (Thermo Fisher Scientific) supplemented with 10% FBS. All cells were maintained in a humidified incubator with 5% CO2 in a constant temperature of 37°C.
To upregulate or downregulate the expression of PUF60, lentivirus containing PUF60 open reading frame (OE-PUF60) and shRNA targeting human PUF60 gene were purchased from OriGene (Beijing, China). And lentivirus (OE-PTEN) used to upregulate PTEN expression was designed and synthesized by GenePharma (Shanghai, China).
To upregulate or downregulate the expression of PUF60, lentivirus containing PUF60 open reading frame (OE-PUF60) and shRNA targeting human PUF60 gene were purchased from OriGene (Beijing, China). And lentivirus (OE-PTEN) used to upregulate PTEN expression was designed and synthesized by GenePharma (Shanghai, China).
5
Culturing TNBC Cell Lines
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Human TNBC cell line MDA-MB-231 (American Type Culture Collection, ATCC) and MDA-MB-231-Luc (expressing luciferase reporter gene) were grown and cultured in DME/F-12 (1:1) medium (Thermo Scientific) containing 10% FBS inside a 37 °C incubator with 5% CO2 and a humidified atmosphere.
6
Establishment of PTX-Resistant Breast Cancer Cell Line
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Human normal breast epithelial cell line MCF10A and human breast cancer cell line MDA-MB-231 were purchased from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). PTX-resistant cell line (MDA-MB-231/PTX) was established by continuous exposure of MDA-MB-231 cells to a stepwise gradually concentration of PTX for more than 3 months, as previously described.16 (link) Cells were maintained in Dulbecco’s Modified Eagle medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific) containing penicillin-streptomycin (Sigma Aldrich, St. Louis, MO, USA), and incubated at 37°C in a humidified atmosphere containing 5% CO2.
7
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.
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.
8
Establishment of Metastatic Breast Cancer Model
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Human mammary carcinoma cell line MDA-MB-231 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and the cells cultured in the Leibovitz L-15 medium supplemented with 10% fetal bovine serum (FBS), streptomycin (100 mg/ml) and penicillin (100 U/ml). The same culture medium, adding 200 μg/ml G418 (Life Technologies, Carlsbad, CA, USA) was also applied to parental MDA-MB-231 (231-P) cells and the metastatic subline of MDA-MB-231 (231-B) cells derived from the bones of our animal models, both of which had stable luciferase expression. All these cells were maintained in the incubator without gas exchange with outside air.
Five-week-old female Balb/c nude mice were purchased from Vital River Laboratories (Beijing, China) and bred in the SPF Animal Center of Cancer Institute and Hospital, Chinese Academy of Medical Sciences. All the animal experiments were approved by the Institutional Review Board of the Chinese Academy of Medical Sciences Cancer Institute.
Five-week-old female Balb/c nude mice were purchased from Vital River Laboratories (Beijing, China) and bred in the SPF Animal Center of Cancer Institute and Hospital, Chinese Academy of Medical Sciences. All the animal experiments were approved by the Institutional Review Board of the Chinese Academy of Medical Sciences Cancer Institute.
9
Cell Lines and Reagents for Cancer Research
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Human breast cancer MDA-MB-231, murine breast cancer 4T1, and endothelial C166 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA). MDA-MB-231-RFP-Luciferase cells were obtained from GenTarget Inc. All cell lines were assessed for mycoplasma contamination using MycoAlert Mycoplasma Detection Kit (Lonza, Walkersville, MD) every 6 months. 4T1 cells were maintained in RPMI1640 supplemented with 10% fetal bovine serum (FBS) and antibiotics. MDA-MB-231 and MDA-MB-231-RFP-Luc cells were cultured in Leibovitz’s L-15 medium supplemented with 10% FBS (Life Technologies, Grand Island, NY). C166 cells were cultured in with DMEM medium supplemented with 10% FBS. Recombinant mouse (rm) and human (rh) CXCL17 was obtained from R&D Systems (Minneapolis, MN, USA). CID 2745687 and DMPQ dihydrocloride (5,7-Dimethoxy-3-(4-pyridinyl)quinoline dihydrochloride, DMPQ 2HCl) were obtained from Torcis (Minneapolis, MN, USA). PDGF-BB was obtained from ProSpec (Ness Ziona, Israel).
10
Cell Culture and Transfection Protocols
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MDA-MB-231, MDA-MB-435, 4T1 and NIH 3T3 cells lines were obtained from the American Type Culture Collection (Manassas, VA, USA). TSA were provided by Dr. P. Lollini, Istituto di Cancerologia, University of Bologna, Italy, and A375M were provided by Dr Daniele Bergamaschi, QMUL, UK. MDA-MB-231 were cultured in DMEM (Life Technologies, UK); MDA-MB-435, 4T1, TSA and A375M were cultured in RPMI 1640 (Life Technologies or PAA, UK) supplemented with 10% (v/v) Foetal Bovine Serum (FBS), Sodium Pyruvate 1% (v/v), 1X L-glutamine/penicillin/streptomycin and 0.1% gentamycin (v/v). NIH 3T3 cells were maintained in DMEM containing 10% donor calf serum. NIH 3T3 cells were transfected with 1 μg of DNA encoding for eGFP-Akt, eGFP-Akt-mRFP or eGFP-PDK129 (link) using Lipofectamine LTX with PLUS reagent (Life Technologies) in OptiMEM containing GlutaMAX (Life Technologies), as recommended by the manufacturer. MDA-MB-231 cells were co-transfected with pOZ-PDK1 and PRK5-PLCγ1 using Lipofectamine (Life Technologies) according to the manufacturer’s instructions. siRNA transfection was performed by using Hiperfect (Qiagen, UK) for A375M as previously described17 (link). SMARTpool siRNA targeting PLCγ1 were purchased from Dharmacon, USA whilst the non-targeting siRNA (Silencer® Negative Control #1 siRNA) was purchased from Applied Biosystems.
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