Egm endothelial cell growth medium bulletkit

All human primary vascular ECs including human aortic ECs (HAECs), human coronary artery ECs (HCAECs), human dermal microvascular ECs (HDMECs), human lung microvascular ECs (HLMECs), and human umbilical vein ECs (HUVECs) were purchased from Lonza Walkersville Inc. maintained in EGM™ Endothelial Cell Growth Medium BulletKit™ or EGM™-2 Endothelial Cell Growth Medium-2 BulletKit™ according to the manufacturer's instruction. For the present study, cells less than passage five were used for the experiment. THP-1 cells (human acute monocytic leukemia cell) were obtained from ATCC and cultured in 10% FBS (Sigma-Aldrich) RPMI 1640 medium (Corning), 1% penicillin/streptomycin (Invitrogen), and 0.05 mmol/L 2-mercaptoethanol (Sigma Aldrich). HEK293T cells were bought from ATCC and seeded in Dulbecco's modified Eagle's medium supplemented with 10% FBS and 1% penicillin/streptomycin (Invitrogen). Transfection of Ataxin-10 vector and siRNA into HUVECs transiently was performed by electroporation using Nucleofector device and Nucleofector kits for HUVEC (Lonza) as described [22 (link), 23 (link)]. After 24 h, 10 ng/mL TNF-α was added to the cells for indicated times, and cell lysates were collected and detected by Western blotting.

Validated stocks of human Jurkat (T-ALL) and Nalm-6 (B-ALL) cell lines were cultured in RPMI1640 media supplemented with 10% FBS. The cell lines were free of mycoplasma contamination. HUVEC (Umbilical Vein Endothelial Cells, CC-2517, Lonza) were cultured in EGM^® Endothelial Cell Growth Medium BulletKit^® (Lonza) supplemented with 1% Penicillin-Streptomycin (5000 U/mL). Human clonal hepatocyte PH5CH8 cells were kindly provided by Dr. Kyle Hoehm, School of Biotechnology & Biomolecular Sciences, UNSW, and cultured in DMEM media supplemented with 10% FBS. The cells were maintained in a humidified atmosphere (5% CO₂) at 37°C and, resuspended in fresh media, and allowed to acclimatize for 24 hrs prior to each experiment.