Cell culture

Cells were maintained in a humidified atmosphere, supplemented with 5.1% CO₂, at 37°C. The non-tumorigenic human breast epithelial cell line MCF-10A (ATCC, Manassas, VA) and its derivatives were grown in DMEM:F12 medium (Life Technologies, Grand Island, NY) devoid of phenol red and supplemented with 5% horse serum (Sigma, Saint Louis, MO), 1% penicillin and streptomycin (Life Technologies, Grand Island, NY), 20 ng/mL epidermal growth factor (EGF; Sigma, Saint Louis, MO), 10 μg/mL bovine serum insulin (Sigma, Saint Louis, MO), 0.5 μg/mL hydrocortisone (Sigma, Saint Louis, MO), and 0.1 μg/mL cholera toxin (Sigma, Saint Louis, MO) (henceforth referred to as “10A supplemented medium”). HER2-overexpressing clones of MCF-10A, referred to as M15, were grown in 10A supplemented medium devoid of EGF and supplemented with 0.4 μg/mL puromycin (Life Technologies, Grand Island, NY; henceforth referred to as “M15 supplemented medium”). BT474 breast cancer cells (ATCC, Manassas, VA) and its derivatives were grown in RPMI-1640 medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (Life Technologies, Grand Island, NY) (henceforth referred to as “BT474 supplemented medium”). HEK-293T cells (ATCC, Manassas, VA) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% FBS. Trastuzumab-resistant BT474 cells were grown in BT474 supplemented medium with the addition of 1.0 μM trastuzumab. HEK-293T cells and parental MCF-10A and BT474 cells were purchased from American Type Culture Collection (Manassas, VA). Trastuzumab resistant BT474 and its respective parent line were a generous gift from Dr. Susan Kane (City of Hope, Duarte, CA). Authenticity of these cells was verified using short tandem repeat analysis at the DNA Services Facility at The University of Illinois at Chicago. Table 1 summarizes the different cellular clones used in this study.

Assays for MCF-10A and M15 cells were conducted in “assay medium,” in which the horse serum in 10A supplemented medium was replaced with 1% charcoal-dextran treated fetal bovine serum (CD-FBS; Fisher Scientific, Pittsburg, PA) and EGF was removed. Assays for BT474 cells were conducted using supplemented BT474 medium. Cells were harvested using TrypLE Express (Life Technologies, Grand Island, NY)