Parental and TamR MCF7 cells were obtained from the Rachel Schiff Lab (Baylor College of Medicine, Houston, TX) [46 (link), 47 (link)]; MDA-MB-453 cells were purchased from ATCC (Manassas, VA).
Mda mb 453 cells
Manufactured by American Type Culture Collection
Sourced in United States
MDA-MB-453 cells are a human breast cancer cell line derived from the metastatic pleural effusion of a female patient. They are adherent, epithelial-like cells that can be used for various cell biology and cancer research applications.
Automatically generated - may contain errors
Lab products found in correlation
Mcf 7 cell, by American Type Culture Collection (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Mfm223 cells, by Merck Group (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
T47d cells, by American Type Culture Collection (1 mentions)
Mem non essential amino acid, by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Matrigel, by Corning (1 mentions)
Mda mb 231 cell, by American Type Culture Collection (1 mentions)
Du145 cells, by American Type Culture Collection (1 mentions)
Sh sy5y cell, by American Type Culture Collection (1 mentions)
4t1 cell, by American Type Culture Collection (1 mentions)
Bt474 cells, by American Type Culture Collection (1 mentions)
Mda mb 436 cells, by American Type Culture Collection (1 mentions)
Rat c6 glioma cells, by American Type Culture Collection (1 mentions)
Sk n be 2 cells, by American Type Culture Collection (1 mentions)
Hek293 cell, by American Type Culture Collection (1 mentions)
6 protocols using mda mb 453 cells
1
Cell Line Acquisition and Origin
2
Breast Cancer Cell Line Protein Extraction
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CAL120 cells were a gift from Elgene Lim (Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia). MFM-223 cells were purchased from Sigma-Aldrich. MDA-MB-453 cells were purchased from ATCC (Manassas, VA, USA). SUM185PE cells were purchased from Asterand Bioscience. Cells were cultured in RPMI-1640 (Gibco) and supplemented with 10% (v/v) FBS (Moregate), 10 μg/ml Actrapid penfill insulin (Clifford Hallam Healthcare) and 20 mM HEPES (Gibco).
For harvesting, cells at 80% confluency were washed twice with ice cold 1 × PBS then lysed with RIPA buffer (0.5% (w/v) sodium deoxycholate, 150 mM NaCl, 1% (v/v) NP40, 50 mM Tris–HCl pH 8.0, 0.1% (w/v) SDS, 10% (v/v) glycerol, 5 mM EDTA and 20 mM NaF), supplemented with 10 μg/ml aprotinin, 1 mM PMSF, 10 μg/ml leupeptin, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate and 2.5 mM β-glycerophosphate prior to use. Lysed cells were collected and clarified by centrifugation at 21,130 × g at 4 °C for 10 min, then the protein concentration was determined using a Pierce BCA protein assay kit (Thermo Scientific) according to the manufacturer’s protocol.
For harvesting, cells at 80% confluency were washed twice with ice cold 1 × PBS then lysed with RIPA buffer (0.5% (w/v) sodium deoxycholate, 150 mM NaCl, 1% (v/v) NP40, 50 mM Tris–HCl pH 8.0, 0.1% (w/v) SDS, 10% (v/v) glycerol, 5 mM EDTA and 20 mM NaF), supplemented with 10 μg/ml aprotinin, 1 mM PMSF, 10 μg/ml leupeptin, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate and 2.5 mM β-glycerophosphate prior to use. Lysed cells were collected and clarified by centrifugation at 21,130 × g at 4 °C for 10 min, then the protein concentration was determined using a Pierce BCA protein assay kit (Thermo Scientific) according to the manufacturer’s protocol.
3
Subcellular Fractionation of Breast Cancer Cells
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T47D cells (ATCC) were cultured in RPMI-1640 supplemented with 10% FBS, 0.5% MEM non-essential amino acids (Sigma), 1 mM HEPES (Sigma), 1 mM sodium pyruvate (Sigma), and 200 U/mL human insulin at 37 °C in 5% CO2. MDA-MB-453 cells (ATCC) were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% FBS. Reverse transfection was performed to deliver siRNAs into cells (3 million cells/15 cm dish for T47D and 5 million cells/15 cm dish for MDA-MB-453; two dishes for each treatment) using Lipofectamine RNAiMAX. Three days later, cells were harvested for subcellular fractionation. Sequences of the siRNAs used in this study are listed in Table S1 .
4
Xenograft Tumor Growth Inhibition
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All in vivo studies were performed under an animal protocol (PRO00007499, PI: Shaomeng Wang) approved by the Institutional Animal Care & Use Committee (IACUC) of the University of Michigan, in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Male CB.17 SCID mice were injected subcutaneously with 5 × 106 MDA-MB-453 cells (ATCC) in 5 mg/ml Matrigel (Corning) for tumor growth. When tumors reached an average volume of 100 mm3, mice were randomized based upon their tumor sizes and assigned to different experimental groups. For PD study, drugs or vehicle control (20% PEG400 + 6% CremophorEL + 74% PBS) were given by intraperitoneal (IP) injection and tumor tissues were harvested at indicated time points for Western blotting analysis. For the in vivo efficacy experiment, drugs or vehicle control (20% PEG400 + 6% CremophorEL + 74% PBS) were administered daily by intraperitoneal (IP) injection. Tumor sizes and animal weights were measured 2 times per week. Tumor volume was calculated as: volume (mm3) = (length × width2)/2.
5
Culturing HER2+ and HER2- Breast Cancer Cells
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The MCF7 cells (HER2(−) human breast adenocarcinoma cell line; ATCC, Manassas, VA) were cultured in Eagle’s minimum essential medium supplemented with 10% foetal bovine serum (FBS), 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 0.01 mg/mL bovine insulin, and 100 U/mL penicillin/streptomycin and maintained in a 37 °C incubator balanced with 5% CO2 and 100% humidity. The MDA-MB-453 cells (HER2(+) human breast metastatic carcinoma cell line; ATCC) were cultured in Leibovitz’s L-15 medium supplemented with 10% FBS, 2 mM L-glutamine, and 100 U/mL penicillin-streptomycin and maintained in a 37 °C incubator without CO2.
6
Cell Culture Conditions for Diverse Cell Lines
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C6 rat glioma cells (ATCC #CCL-107), HEK293 cells (ATCC #CRL-1573), GT1-7 cells [62 (link)], CLU188 cells [63 (link)], SH-SY5Y cells (ATCC #CRL-2266), SK-N-BE2 cells (ATCC #CRL-2271), iNHA cells [63 (link)], MDA-MB-231 cells (ATCC #HTB-26), MDA-MB-436 cells (ATCC #HTB-130), MDA-MB-453 cells (ATCC #HTB-131), MCF7 cells (ATCC #HTB-22), 4T1 cells (ATCC #CRL-2539), BT474 cells (ATCC #HTB-20), CHO cells (ATCC #CCL-61), OVK18 cells [64 (link)], DU145 cells (ATCC #HTB-81), and HCCLM3 cells [65 (link)] were maintained as a monolayer culture on tissue culture dishes at 37 °C, 5% CO2, 100% humidity in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum and antibiotics. SH-SY5Y cells were differentiated in the presence of 10 μM retinoic acid for 1 week [66 (link)]. OC-k3 cells [67 (link)] were maintained as a monolayer on tissue culture dishes at 33 °C, 10% CO2, 100% humidity in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 50 U/mL of recombinant mouse interferon-γ, and antibiotics. S1P and CYM-5478 were solubilized with bovine serum albumin (0.1% final concentration) prior to treatment.
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