Hmec 1

Human prostate carcinoma cell line PC-3 (ATCC-CRL-1435), non-small cell lung cancer cell line H157 (ATCC-CRL-5802), breast cancer cell line MDA-MB-435s (ATCC-HTB-129), neuronal glioblastoma cell line U251MG (ECACC-09063001) and breast cancer non-tumorigenic mammary gland cell line MCF-7 (ATCC-HTB-22) were utilized to screen the cytotoxicity of synthetic peptides. PC-3 and H157 cell lines were cultured in RPMI-1640 medium (Invitrogen, Paisley, UK), and the other cell lines were cultured with Dulbecco’s Modified Eagle’s medium (DMEM) (Sigma, St. Louis, MO, USA). Culture media was supplemented with 10% fetal bovine serum (FBS) (Sigma, Welwyn Garden City, UK) and 1% penicillin streptomycin solution (Sigma, UK). The human mammary epithelial cell line, HMEC-1 (ATCC-CRL-3243) was used to evaluate the cytotoxicity of the synthetic peptides against normal human cells, and cultured with MCDB131 medium (Gibco, Paisley, UK) with 10% FBS, 10ng/ml EGF, 10mM L-Glutamine and 1% penicillin streptomycin. The selected cell lines were resuscitated and transferred into a 75 cm² culture flask, and then incubated at 37 °C atmosphere containing 5% CO₂.

Human bone marrow stromal cells (HS27a), human dermal microvascular endothelial cells (HMEC-1), human lung microvascular endothelial cells (HULEC-5a), and PC3 PCa cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Bone marrow microvascular endothelial cells (BMEC-1) [13 (link)] were a gift from Dr. Carlton Cooper (University of Delaware), 22Rv1 prostate cancer cells [14 (link)] were a gift from Dr. Neil Bhowmick (Cedars Sinai, Los Angeles), and C4-2B PCa cells [15 (link)] were a gift from Dr. Leland Chung (at that time at the University of Texas MD Anderson Cancer Center). HS27a, 22Rv1, PC3, and C4-2B were cultured in RPMI 1640 (Gibco, Invitrogen, Carlsbad, CA, USA)) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Atlanta Biologicals, Inc., Flowery Branch, GA, USA) and 1X penicillin–streptomycin (Gibco). HMEC-1 and HULEC-5a were cultured in MCDB131 (Gibco) supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 10 mM L-glutamine (Gibco), 10 ng/mL Epidermal Growth Factor (Gibco), and 1 µg/mL hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA). BMEC-1 cells were cultured in Medium 199 (HEPES, no L-glutamine from Sigma) supplemented with 15% (v/v) heat-inactivated fetal bovine serum, 10 mM L-glutamine, 1X penicillin–streptomycin, 40 ug/mL endothelial cell growth supplement (ECGS, Sigma), and 16 U/mL heparin (Sigma).

Human dermal primary LEC and human primary dermal fibroblasts (HDF) were from PromoCell (Heidelberg, Germany). The HMEC-1 cell line was obtained from the Centre for Disease Control and Prevention (CDC; Atlanta, Georgia, USA). LEC and HDF were cultured in MV2 and FGM2, respectively (both PromoCell). HMEC-1 were cultured in M199 (Gibco, Paisley, UK) that was supplemented with 20% foetal calf serum (Sigma, Poole, UK). With the exception of siRNA transfections, cells were treated with 2.5µg/ml amphotericin (Gibco), 100U/ml penicillin and 100µg/ml streptomycin (both Sigma). All cells were cultured at 37°C and 5% CO₂ and 95% air.

HUVECs and human dermal microvascular endothelial cells (HMEC-1) were obtained from the American Type Culture Collection. Monolayers were cultured in medium 199 (Gibco, Thermo Fisher Scientific) supplemented with 20% heat-inactivated FBS (Gibco, Thermo Fisher Scientific), endothelial cell growth supplement (0.03 mg/mL), heparin (0.1 mg/mL), penicillin (100 IU/mL), and streptomycin (100 μg/mL). HMEC-1 monolayers were cultured in MCDB131 (Gibco, Thermo Fisher Scientific) supplemented with 15% heat-inactivated FBS (Gibco, Thermo Fisher Scientific), endothelial cell growth supplement (20 μg/mL), hydrocortisone (1 μg/mL), and glutamine (10 mM) in a humidified incubator at 37°C and 5% CO₂/95% air.