Bt474

Human breast cancer cell lines BT474, SK-BR-3, MDA-MB-361 and MDA-MB-453 were purchased from the American Type Culture Collection (ATCC) at the beginning of the project in August 2018. Cell lines were cultured in DMEM (Sigma #D6429) supplemented with 10% FBS (Sigma #10500-064) and 1% penicillin/streptomycin (Sigma #P4333), except MDA-MB-361 cells which were grown in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, 2 mM L-glutamine (Sigma #G7513) and 1x MEM non-essential amino acid solution (Sigma #M7145). Lapatinib-resistant (LR) and trastuzumab resistant (TR) BT474 cell lines were developed by incubation with gradually increased doses of lapatinib (APExBIO #A8218) or trastuzumab (Genentech) over a period of 4 to 5 months. Resistant cells were subsequently maintained with 0.1 μM lapatinib or 50 μg/ml trastuzumab. The compounds were removed from the culture medium three to four days prior to experiments. Cell authentication was not routinely conducted given that cell lines were utilized for a maximum of 20 passages before thawing another aliquot of the same stock to maintain the original phenotype. Mycoplasma testing was not routinely performed on the cells. Trametinib was purchased from APExBIO (#A3887). For in vivo studies, lapatinib was dissolved in a buffer containing 1% Tween-80 and 5% hydroxypropyl methylcellulose just before use.