Stable Transfection of Breast Cancer Cell Lines

Human breast cancer cell lines, MDA-MB-231, SUM149, SUM159, BT-474, T-47D, and MCF-7, were obtained from the American Type Culture Collection (ATCC, MD, USA). Cells were stably transfected with a construct containing cDNA of tdTomato as outlined in the Supplementary Materials and Methods under ‘Cell lines’.^{27 (link)} Human mammary fibroblasts were a kind gift from Dr Gary Luker at the University of Michigan. All cells were incubated at 37 °C with 5% CO₂ in a humidified incubator.
Descriptions of cell-specific culture media are given in Supplementary Materials and Methods under ‘Cell type specific media used for breast cancer cell lines’. tdTomato protein expression was detected by fluorescence microscopy using a ×20 objective attached to a Nikon inverted microscope, equipped with a filter set for 528 to 553 nm excitation and 600 to 660 nm emission and a Nikon COOLPIX digital camera (Nikon Instruments, Inc, Melville, NY, USA).^{27 (link)}