Microvascular endothelial cell growth medium 2 bullet kit

Primary human umbilical vein endothelial cells (HUVECs) (ScienCell, Carlsbad, CA, USA; Cat.#: 8000) were cultured in endothelial cell growth media (Endothelial Cell Growth Medium‐2 BulletKit, Lonza, Cat.#: CC‐3162) and primary human retinal microvascular endothelial cells (HRMECs) (Cell Systems, Kirkland, WA, USA, Cat. #ACBRI 181) were cultured in microvascular endothelial cell growth medium (Microvascular Endothelial Cell Growth Medium‐2 BulletKit, Lonza, Cat.#: CC‐3202) at 37 °C in a humidified atmosphere containing 5% CO₂.

Human PC3, DU145 and LNCaP cell lines were obtained from ATCC (Manassas, VA) and maintained in DMEM High Glucose (PC3 and DU145) or RPMI-1640 (LNCaP) (HyClone) with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin in 5% CO₂ humidified atmosphere at 37°C. Primary antibodies against cleaved caspase-3, cleaved caspase-9, N-Cadherin, p-GSK3 Ser9/21, β-catenin and Snail were purchased from Cell Signaling (Boston, MA), and anti-β-actin was from Sigma (St Louis, MO). Anti-mouse and anti-rabbit HRP conjugated secondary antibodies were obtained from BioRad (Hercules, CA). Human dermal microvascular endothelial cells (HMEC) were obtained from ATCC. Cells were maintained in Endothelial Cell Basal Medium-2 (Lonza) with Microvascular Endothelial Cell Growth Medium-2 Bullet Kit (Lonza). All cultures were maintained in a humidified 5% CO2 incubator at 37°C, and routinely passaged when 80–90% confluent.

Human retinal endothelial cells (HRECs) were procured from Cell Systems (Kirkland, WA, USA) and cultured, as previously described^{38 (link),39 (link)}, in Microvascular Endothelial Cell Growth Medium-2 Bullet Kit (Lonza, Walkersville, MD, USA; Catalog No. CC-3202 EGM-2 MV), which includes base medium and growth kit supplements, containing human fibroblast growth factor (hFGF), human vascular endothelial growth factor (hVEGF), human insulin-like growth factor (hIGF-1), human epidermal growth factor (hEGF), and 5% fetal bovine serum (FBS). HRECs of passage 3–6 were seeded in 100 mm Petri dishes until they reached 90% confluency, then the medium was changed to fresh medium containing 5% FBS, but without growth factors, that had either mannitol (25 mM), which was the osmotic control group, or HG (25 mM) for 5 days. Hypoxia (Hyp) was introduced 24 h before cell harvesting by incubating HRECs in a 2% O₂ humidified hypoxic chamber with concurrent 5% CO₂.