Establishing Lapatinib-Resistant Breast Cancer Cell Lines

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).