Megm complete medium

U2Os, U2Os-AP2-GFP, U2Os-AP2-halo, U2Os-AP2-GFP-ITGB5-mScarlet, HeLa-AP2-GFP, and HeLa-AP2-halo were cultured in MEM supplemented with 10% FBS (Gibco) and penicillin–streptomycin (100 U/ml, Thermo Fisher Scientific). HDF-AP2-halo and Caco2-AP2-halo were cultured in DMEM supplemented with 10% FBS (Gibco) and penicillin–streptomycin (100 U/ml, Thermo Fisher Scientific). MCF7-AP2-halo was cultured in DMEM supplemented with 10% FBS (Gibco) and penicillin–streptomycin (100 U/ml, Thermo Fisher Scientific), 2 mM glutamine, 5 µg/ml human insulin, and 1 µM sodium pyruvate. hMEC-AP2-GFP was cultured in MEGM complete medium (Lonza).
The following primary antibodies were used: anti-human integrin β1 clones 12G10 (NB100-63255; Novus bio), mAb13 (552828; BD), total integrin β1 (MAB2252; Millipore), anti-human integrin αvβ5 clone 15F11 (MAB2019Z; Millipore), anti-human integrin α5 clone SNAKA51 (AF1846; R&D), anti-human Tensin1 (SAB4200283; Sigma-Aldrich), anti-human p-paxillin Y118 (69363; Cell Signaling), anti-human-Talin1 (T3287; Sigma-Aldrich), anti-α-tubulin (sc-32293; SantaCruz Biotechnology), and anti-GAPDH (G9545; Sigma-Aldrich). Corresponding secondary antibodies raised against rabbit or mouse IgG were purchased from Jackson Immunoresearch.

Human breast cancer cell lines were purchased from ATCC (Manassas, VA) and most cell experiments were performed within 6 months after their receipt. However, after the completion of experiments outlined in this paper, the identity of all cell lines used in this study was confirmed by the Human Cell Line Authentication test (Genetica DNA Laboratories, Burlington, NC). Cells were cultured in a base medium supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies), 50 units/mL penicillin, and 50 μg/mL streptomycin. The base medium for MDA-MB-231 was DMEM, for HCC1954 was RPMI, and for MCF-7 cells was insulin-supplemented MEM (all from Fisher Scientific, Pittsburgh, PA). MCF-10A cells were grown in MEGM complete medium (Lonza, Basel, Switzerland) that was replaced by RPMI containing 10% FBS 24 hours prior to treatment with blood samples. Serum, plasma, purified IgG or mAbs were used to treat cells. MAbs used in our studies include the LeY reactive MAb BR55–2 (a gift from Zenon Steplewski [74 (link)]), 14G2a (BD Biosciences, San Jose, CA) and 3F8 (a gift from Dr. Ni-Kong V. Cheung, Memorial Sloan Kettering Cancer Center, New York, NY).