Heregulin 1β epidermal growth factor like domain

All human breast cancer cell lines (MCF7, T47D, HCC1428, MDA-MB-361) were obtained from American Tissue Type Collection (Rockville, MD) and were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologics) except where indicated as serum-free. Fulvestrant (1 μM) (Sigma-Aldrich) and Heregulin-1β epidermal growth factor-like domain (2 ng/ml, R&D Systems), and the humanized ErbB3 monoclonal antibody A4 ^{14 (link),15 (link)} were used where indicated. Colony assays were seeded in 6-well dishes (10⁵ cells/dish), and cultured 14d in DMEM/10% FBS. Cells were fixed in formalin, stained with crystal violet, imaged on a flatbed scanner, converted to binary images, and quantified using Image J software. Cells (1 X 10⁵) were transfected using Xfect Reagent (Clontech) according to manufacturer’s directions with 2 μg plasmid DNA: pcDNA3.1 (Life Technologies/InVitrogen), pcDNA3.1-LRIG1, pcDNA3.1-LRIG1-ΔEcto, and pcDNA3.1-LRIG1-ΔCyto^{21 (link),22 (link)}. LRIG1 siRNA and non-specific siRNA sequences were purchased (Dharmacon) and used for transfection of cells with Dharmafect (Dharmacon) according to manufacturer’s directions.