Hmecs

Human normal mammary epithelial cells (HMECs) were obtained from Lonza Group (San Diego, CA, USA). Cells between passages 10 and 11 were used for experiments and the cells were grown and maintained in mammary epithelial basal medium supplemented with 13 mg/ml bovine pituitary extract, 0.5% serum, 5 μg/ml insulin, 10 ng/ml human recombinant epidermal growth factor, 0.5 mg/ml hydrocortisone, 50 μg/ml gentamicin and 50 μg/ml amphotericin-β (all Clonetics, Lonza Group, San Diego, CA, USA). Cells were maintained in a humidified environment at 37°C with 5% CO₂ in air.
Four different human breast cancer cell lines (MCF-7, T47D, MDA-MB-231 and MBA-MB-435) purchased from the American Type Culture Collection (Manassas, VA, USA) were grown and maintained in appropriate growth media; minimal essential medium for MCF-7 and RPMI 1640 for T47D, MDA-MB-231 and MBA-MB-435 (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Welgene, Daegu, Korea). L-glutamine, penicillin, streptomycin and gentamicin (Life Technologies Korea, LLC, Seoul, Korea) were used at the usual concentrations. For all experiments, breast cancer cells were harvested by trypsinization (0.25% trypsin and 0.02% EDTA; Life Technologies Korea, LLC), seeded and grown in the appropriate media containing 10% FBS in a humidified 95% air 5% CO₂ atmosphere.

Human breast adenocarcinoma cell lines (MDA-MB-231, MDA-MB-468, and BT-549), HMECs (MCF-10A) and luciferase-expressing MDA-MB-231 cells (MDA-MB-231-Red-FLuc Bioware® Brite) were obtained from ATCC (Manassas, VA, USA) and PerkinElmer (Street Waltham, MA, USA), respectively. All cell lines were authenticated by short tandem-repeat profiling and cell morphology assessment. BT-549 cells were maintained in DMEM (Gibco, Grand Island, NY, USA) with 10% FBS, and 0.01 mg/ml human recombinant insulin. MDA-MB-468 cells were maintained in L-15 medium (Gibco) with 10% FBS. All MDA-MB-231 cell lines, including MDA-MB231-FLuc, MDA-MB-231-RAGE^OV and MDA-MB-231-RAGE^KD, were cultured in RPMI 1640 medium (Gibco) with 10% FBS^{12 (link)}. HMECs and HUVECs (Lonza Group Ltd., Walkersville, MD, USA) were cultured in specific epithelial and endothelial growth medium (Lonza), respectively.

HMECs were obtained from Lonza and cultured according to the manufacturer’s guidelines. Other cell lines were obtained from ATCC and cultured in the following media: MDA-MB-231: DMEM, 10% FBS; MDA-MB-361: DMEM, 20% FBS; BT549: RPMI-1640, 10% FBS, 0.001 mg/ml bovine insulin; MCF7: DMEM, 10% FBS, 0.01 mg/ml bovine insulin; HEK293T: the same medium as NMuMG. HMLE cells were a kind gift from Christina Scheel (Helmholtz Zentrum Munich) and cultured in LONZA primary cell culture medium. A subclone of NMuMG cells was grown in DMEM supplemented with 10% FBS, 2 mM L-glutamine and 1× non-essential amino acids^{62 (link)}. All the cells were cultured at 37 °C with 7% CO₂ in a humid incubator. For TGF-β time-course experiments, NMuMG, HMEC and HMLE cells were treated with 2, 5 and 5 ng/ml TGF-β, respectively, for the indicated times. TGF-β was replenished and the medium was changed every 2 days.

Human dermal microvascular endothelial cells (HMECs; Lonza, Basel, Switzerland) were cultured as described previously (Rydkina et al., 2010 (link)). Briefly, cells were cultured in MCDB 131 medium (Caisson’s Laboratories) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Shanghai, China), 10 ng/ml epidermal growth factor (Thermo Fisher Scientific), 1 μg/ml hydrocortisone (Sigma-Aldrich, Shanghai, China), and 10 mM L-Glutamine (Thermo Fisher Scientific). LPS from Escherichia coli (E. coli O111: B4; Invivogen, San Diego, CA, United States) was resuspended in distilled water just before use. HMECs were treated with 1 μg/ml LPS for 24 h.

MCF-7, ZR-75-1 and MDA-MB-231 cell lines were obtained from American Type Culture
Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco’s modified
essential medium supplemented with 10% FBS and antibiotics. HMECs were
obtained from LONZA (MD, USA) and maintained in MEBM (mammary epithelial basal
medium) with growth factors. Cell lines were grown in a humidified incubator with
5% CO₂ at 37 °C. The cell lines used in these studies
were authenticated as a standard quality control measure at the beginning and end
of the project and when banking frozen materials for later use. The
authentications of these cells were performed using STR profiling (Short Tandem
Repeat analysis of DNA) and were checked every two months for mycoplasma
contamination.
Transfection was performed with the Neon Transfection System (Thermo Fisher
Scientific, Waltham, MA, USA) as per manufacturer’s instruction. Briefly,
~70% confluent cells were trypsinized, washed with 1 × PBS and then
suspended in Buffer R and mixed with plasmid DNA (5–10 μg). The
cell-DNA mixtures were electroporated for transfection at selected voltage pulse
using Neon Transfection System.

HMECs (Lonza, Basel, Switzerland) were cultured in endothelial cell medium with 5% foetal bovine serum, 1% endothelial cell growth supplement, and 1% penicillin/streptomycin solution (ScienCell Research Laboratories, Carlsbad, USA) at 37 °C under a 5% CO₂ atmosphere. HMECs between passages 3–7 were used in this study. Cells were treated with or without ox-LDL for 1 h followed by treatment with 100 μg/mL ECFC-exosomes or an autophagy inhibitor (10 mM bafilomycin A1, Selleckchem) for another 24 h. In the ECFC-exosome treatment experiments, HMECs were cultured in MCDB131 medium without serum or growth factors.