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2 protocols using lapatinib gw 572016

1

Evaluation of Anti-Cancer Agents in HER2-Positive Cell Lines

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BT474, HCC1954, and MDA-MB 361 cells were obtained from the American Type Culture Collection (ATCC) between 2006–2011 and maintained in ATCC-recommended media supplemented with 10% FBS (Gibco) and 1x antibiotic/antimycotic (Gibco). All cell lines were authenticated by ATCC using the short tandem repeat (STR) method in January 2017. Mycoplasma testing was conducted for each cell line before use. All experiments were performed less than 2 months after thawing early passage cells.
The following drugs were used: lapatinib (GW-572016, LC Laboratories), trastuzumab (Vanderbilt University Hospital Pharmacy), lucitanib (14 (link)) (provided by Clovis Oncology), and AZD4547 (15 (link)) (provided by AstraZeneca Pharmaceuticals).
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2

Establishing Lapatinib-Resistant Breast Cancer Cell Lines

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ErbB2+ (BT474; SKBR3) and ErbB2 negative (MCF-7; T47D) human breast cancer cell lines, and the human foreskin fibroblast (HFF) cell line were obtained from the American Type Culture Collection (Manassas, VA). Lapatinib resistant breast cancer cells (rBT474; rSKBR3) were generated and maintained in culture as previously described [16] (link)–[18] (link). Cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum and L-glutamine from GIBCO (Grand Island, NY) growing in a humidified atmosphere of 5% CO2 at 37°C. IRDye 800 conjugated affinity purified anti-rabbit IgG and anti-mouse IgG were from Rockland (Gilbertsville, PA). Alexa Fluor 680 goat anti-rabbit IgG were purchased from Molecular Probes (Eugene, OR). Anti-PARP (Poly (ADP-ribose) Polymerase) monoclonal antibody was from BD PharMingen (San Jose, CA). 8-Methoxypsoralen (8MOP) and the 4G10 anti-phosphotyrosine (p-tyr) antibody were purchased from Sigma-Aldrich (St. Louis, MO). Monoclonal antibodies to c-ErbB2 and EGFR were purchased from Neo Markers (Union City, CA). PARP cleavage product was obtained from Cell Signaling (Beverly, MA). Antibodies to Akt1/2, phospho-Akt1/2 (S473), phospho-Akt1/2 (T308), phospho-Erk1/2 and Erk1/2 were purchased from Santa Cruz (Santa Cruz, CA). Lapatinib (GW572016) and neratinib (HKI-272) [19] were purchased from LC Laboratories (Woburn, CA).
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