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7 protocols using bt 474 htb 20

1

Anticancer Compound Screening Protocol

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Fulvestrant and sorafenib were purchased from AK Scientific (Mountain
View, CA); crizotinib from Selleck Chemicals (Houston, TX); rosmarinic
acid, donepezil, and rosiglitazone from Santa Cruz Biotechnology (Dallas,
TX); ketotifen, verapamil, genistein, and GLP-1R agonist from Sigma-Aldrich
(St. Louis, MO); benzoquinoline from Spectrum Chemicals (New Brunswick,
NJ); AFP 07 from Caymen Chemical (Ann Arbor, MI); phenol red from
Amresco (Solon, OH); tetrahydroberberine from Vitas-M (Moscow, Russia).
Ultrapure polysorbate 80 (UP 80) was purchased from NOF Corporation
(White Plains, NY). Dulbecco’s phosphate buffered saline (DPBS)
and RPMI 1640 cell culture media were purchased from Multicell Technologies
(Woonsocket, RI). Charcoal-stripped Fetal bovine serum (FBS) was purchased
from Sigma-Aldrich (St. Louis, MO). Cell lines MDA-MB-231 (HTB-26),
MCF-7 (HTB-22), SK-BR-3 (HTB-30), and BT-474 (HTB-20) were purchased
from ATCC (Manassas, VI). MTS cell proliferation assay was purchased
from Promega (Madison, WI). Duke Standards NIST Traceable Polymer
Microspheres were purchased from Thermo Scientific. FluoSpheres fluorescent
nanoparticles were purchased form Life Technologies (Burlington, ON).
All other chemicals and reagents were purchased from Sigma-Aldrich
(St. Louis, MO) or TCI America (Portland, OR).
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2

Comprehensive Breast Cancer Cell Line Panel

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Human normal breast epithelial cell line (MCF-10A, CL-0525) and BC cell lines (MDA-MB-231 (CL-0150), SK-BR-3 (SKBR-3, CL-0211), MCF-7 (CL-0149), HS-578T (CL-0114), MDA-MB-468 (CL-0290), T-47D (CL-0228), and BT-474 (CL-0040)) were obtained from Procell (Wuhan, China); BC cell lines (HCC1806 (CRL-2335) and BT-474 (HTB-20)) were acquired from ATCC (Rockville, USA) and cultivated in the recommended media. All cell lines were subcultured in our laboratory for less than six months.
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3

Cell Culture Protocols for Cancer Studies

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Murine breast cancer 4T1 (#CRL-2539) and LLC1 (#CRL-1642), colon cancer (CT26) (#CRL-2638), and human MCF-7 (#HTB-22), BT-474 (#HTB-20), and MDA-MB-231 (#HTB-26) breast cancer cells were purchased from American Type Culture Collection, USA. 4T1-12B cells were a gift from G. Sahagian, Tufts University, USA. 4T1 and 4T12B cells were cultured in RPMI-1640, and LLC1, MCF-7, MDA-MB-231, and BT-474 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) and 1× penicillin-streptomycin solution at 37°C and 5% CO2. Cells were grown to 70 to 80% confluency and harvested using 1× trypsin.
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4

Generating Lapatinib-Resistant Breast Cancer Cell Lines

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Human SkBr3 (HTB‐30) and BT474 (HTB‐20) breast cancer cell lines were obtained from the American Type Culture Collection (ATCC). Lapatinib‐resistant (LR) clones were generated from long‐term treatment with Lapatinib (GlaxoSmithKline).24 (link), 25 (link) All cell lines were cultured in Dulbecco's modified Eagle medium/F12 (SH30004.04, HyClone) supplemented with 10% fetal bovine serum (10437‐028, Gibco), 1% penicillin‐streptomycin (SH40003.01, HyClone) at 37°C in an humidified atmosphere of 95% air and 5% CO2. LR clones were maintained in the presence of 1 μmol/L Lapatinib.
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5

Characterization of HER2+ Breast Cancer Cell Lines

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HER2+ breast cancer cell lines BT474 (HTB-20) representing Luminal B (ER+; PR+, HER2+; ductal carcinoma) subtype and SKBR3 (HTB-30) representing HER2+ subtype (ER-; PR-; HER2+; adenocarcinoma) of breast cancer were purchased from the American Type Culture Collection (ATCC) [25 (link),26 (link)], which were known to well characterized in previous studies and decribed highly sensitive to trastuzumab treatment [27 (link),28 ]. SKBR3 cells were maintained in Mc Coy’s 5A medium (Lonza) with L-glutamine containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin. BT474 cells were maintained in RPMI 1640 medium with L-glutamine (Lonza) supplemented with 10% FBS, 1% penicillin-streptomycin and 2% bovine insulin. The cell lines were cultured in a humidified air supplemented with 5% CO2 at 37°C. Trastuzumab was (kindly gifted by Prof. Dr. Hakan Gürdal from the Department of Pharmacology in Medical School of Ankara University) dissolved in phosphate-buffered saline (PBS) (stock concentration of 300 mg/mL) and stored at 4°C.
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6

Culturing Human and Mouse Cell Lines

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The human epidermis carcinoma cell line A431 (ECACC85090402) was obtained from the European Collection of Authenticated Cell Cultures through DS Pharma Biomedical (Osaka, Japan). The mouse embryo fibroblast cell line BALB/3T3 clone A31 (JCRB9005) was obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). The human breast ductal carcinoma cell line BT-474 (HTB-20) was obtained from the American Type Culture Collection through Summit Pharmaceuticals International Corporation (Tokyo, Japan). These cell lines were cultured in Dulbecco’s modified Eagle’s medium (10569010; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (26140079; Thermo Fisher Scientific) and 1% penicillin/streptomycin (15140122; Thermo Fisher Scientific). Each cell line was cultured in tissue culture flasks at 37 °C in a 5% CO2 humidified atmosphere.
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7

Breast Cancer Cell Line Cultivation

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Breast cancer cell lines MCF-7 (TCHu74), MDA-MB-231 (TCHu104) were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences. BT474 (HTB-20™), and T-47D (HTB-133) and normal mammary epithelial cell line MCF 10A (CRL-10317) were obtained from the American Type Culture Collection (Manassas, VA, USA). The cell identity was confirmed by STR analysis. The MCF-7, BT474, MDA-MB-231 and T-47D cells were cultured in Dulbecco's modified Eagle's medium (DMEM; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone; GE Healthcare Life Sciences). The MCF-10A cells were cultured in DMEM/F12 medium (HyClone; GE Healthcare Life Sciences) containing 5% horse serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 1% penicillin/streptomycin (HyClone; GE Healthcare Life Sciences), 20 ng/ml of EGF (Gibco; Thermo Fisher Scientific, Inc.), 0.5 µg/ml of hydrocortisone (Sigma, St. Louis, MO, USA). All cells were incubated in a humidified atmosphere with 5% CO2 and humidified sphere of 95% at 37°C.
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