Culturing and Engineered Cell Lines

Human mammary epithelial cells (HMLE) were obtained from Sendurai A. Mani (MD Anderson Cancer Center, Houston TX) and NMuMG and BT474 cells were purchased from the ATCC (Manassas, VA, USA). The HMLE cells were cultured in DMEM:F12 supplemented with insulin (10 μg/ml), EGF (10 ng/ml), and hydrocortisone (250 μg/ml), this media was mixed 1:1 with Mammary Epithelial Cell Growth Medium (MEGM) purchased from Lonza (Allendale, NJ, USA). Bioluminescent HMLE cells were engineered to stably express firefly luciferase via lentiviral transduction under the selection of Blasticidin. Her2 and Twist expressing HMLE cells and Twist expressing NMuMG cells were constructed via stable transduction using pBabe viral particles and selected for using puromycin. NMuMG, Her2 transformed HMLE and BT474 cells were cultured in DMEM supplemented with 10% FBS, 1% Pen/Strep, and 10 μg/mL of insulin. Plasmids encoding eGFP, FGFR1-α-IIIc (NM_023110.2) or FGFR1-β-IIIc (NM_023105.2) were purchased from Cyagen Biosciences (Santa Clara, CA, USA). These constructs were used to construct lentiviral particles, and stable transduction was selected for under Hygromycin selection. TGF-β1 and basic FGF (FGF2) were purchased from R&D systems (Minneapolis, MN). BGJ-398, Lapatinib, Afatinib, Trametinib, VX11e, AZD-6244, and Idelalisib were purchased from Selleckchem (Houston, TX), solubilized in DMSO and used at the indicated concentrations. FIIN-2 and FIIN-4 were synthesized as previously described and similarly solubilized in DMSO [15 (link), 18 (link)].