Supplementary MaterialsAdditional file 1: Figure S1. FBS (HyClone, Logan, UT), and penicillin 100?U/ml/streptomycin 100?g/ml (Gibco). Twenty-four hours later, the medium was replaced with 1 differentiation medium (MEM, 10% neonatal FBS, penicillin 100?U/ml/streptomycin 100?g/ml, 50?g/ml ascorbic acid (Sigma, St. Louis, MO), and 10?mM -glycerophosphate (Sigma, St. Louis, MO)). MC3T3-E1 cells were grown to two stages of differentiation: early differentiation (10?days) or late differentiation (20?days) and were used at passage ?20 [41]. Differentiation medium was exchanged every third day. Cells were cultured in a humidified chamber of 5% CO2 and Ridinilazole 95% air at 37?C. NHOst human primary osteoblasts derived from a single donor with no evidence of disease were purchased directly from Lonza (Walkersville, MD). NHOst cells were maintained in a growth media of osteoblast basal medium plus FBS, ascorbic acid, and gentamicin/amphotericin-B (Lonza). Media were exchanged every other day. Cells were cultured in a humidified chamber of 5% CO2 and 95% air at 37?C. Mouse fibroblastsNIH-3T3 murine fibroblast cells are a mesenchymal cell line established from NIH Swiss mouse primary embryo cultures [42]. These cells were a gift from Dr. Andrea Mastro, The Pennsylvania State University. Media were exchanged every other day. NIH-3T3 cells were maintained in a growth media of alpha-MEM (Gibco), 10% FBS (Hyclone), and penicillin 100?U/ml/streptomycin 100?g/ml (Gibco). Cells were cultured in a humidified chamber of 5% CO2 and 95% air at 37?C. Human mammary epithelial cellshTERT-HME1 human mammary epithelial cells were derived from a patient undergoing reduction mammoplasty with no history of breast cancer. The human mammary epithelial cells were immortalized by infection with pBabepuro-hTERT vector retrovirus [43]. hTERT-HME1 cells were maintained in mammary epithelial cell growth medium (MEBM) supplemented with bovine pituitary extract, hydrocortisone, human epidermal growth factor (10?g/ml), 0.5% recombinant human insulin, and gentamicin/amphotericin-B (Lonza). hTERT-HME1 cells were purchased from the ATCC (Manassas, VA). Cells were cultured in a humidified chamber of 5% CO2 and 95% air at 37?C. Cancer cellsMDA-MB-231 human metastatic breast cancer cells were derived from a pleural effusion of an adenocarcinoma [44]. MDA-MB-231BRMS human metastasis-suppressed cells are the isologous line in which metastasis is suppressed to the bone as well as to the other organs by transfection of the BRMS1 gene [45, 46] and were a gift from Dr. Danny Welch, Kansas University Medical Center. MDA-MB-231 and MDA-MB-231BRMS cells were maintained in a breast cancer growth medium of DMEM (Gibco), 5% neonatal FBS, and 1% penicillin 100?U/ml/streptomycin 100?g/ml. Cells were cultured in a humidified chamber of 5% CO2 and 95% Rabbit polyclonal to Complement C4 beta chain air at 37?C. MCF-7 human ER+ breast cancer cells were derived from a Ridinilazole pleural effusion [47] and were purchased directly from the ATCC (Manassas, VA). MCF-7 cells were maintained in EMEM (Gibco) supplemented with 10% FBS (Hyclone), 100?U/ml penicillin 100?mg/ml streptomycin (Gibco), and 0.01?g/ml of recombinant human insulin (MP Biomedicals, Solon, OH). For in vivo experiments, cell lines expressing the green fluorescent protein (GFP) and luciferase (pLeGo-IG2-Luc2 vector) were utilized and were a gift from Dr. Alessandro Fatatis, Drexel University. MDA-MB-231GFP/luciferase cells are analogous to MDA-MB-231 cells but have been engineered to express GFP and the Luc2 vector [48]. Conditioned media MC3T3-E1 cells, grown for 10 or 20?days, were rinsed with phosphate-buffered saline (PBS) and serum-free MEM added (20?ml per T-150 flask, ~?9.1??104 cells/cm2) for 24?h. Osteoblast-conditioned medium (OBCM) was collected, centrifuged to remove cellular debris, and stored at ??80?C. MDA-MB-231 triple-negative metastatic breast cancer, Ridinilazole MDA-MB-231BRMS metastasis-suppressed breast cancer cells, MCF-7 ER+ breast cancer cells, or hTERT-HME1 human mammary epithelial cells were rinsed with PBS and serum-free MEM added (~?1.3??105 cells/cm2). Twenty-four hours later, breast cancer cell-conditioned medium (BCCM) or hTERT-HME1-conditioned medium was collected, centrifuged to remove cellular debris, and stored at ??80?C. Generation of EOs in vitro Differentiated MC3T3-E1 cells were rinsed and treated with either BCCM or hTERT-HME1-conditioned media treatment formulation: 3 parts 1.5 differentiation medium (MEM, 15% neonatal FBS, 75?g/ml ascorbic acid (Sigma), 15?mM -glycerophosphate (Sigma), and penicillin 100?U/ml/streptomycin 100?g/ml) plus 1 part either MDA-MB-231, MDA-MB-231BRMS, or MCF-7 breast cancer-conditioned medium; or hTERT-HME1 mammary epithelial cell-conditioned medium for an additional 21?days [49] (days 31 or 41, respectively). Media were changed every second day. Vehicle medium (VM) consisting of.