Human umbilical vein endothelial cells (huvec)

Human cervical cancer cell line HeLa (adenocarcinoma from ATCC, Manassas, VA) and benign human bladder cell line, UROtsa (a generous gift from Dr. Donald Sens at the University of North Dakota School of Medicine, Grand Forks, ND) were available for analysis. HeLa cells were maintained in RPMI 1640 media and UROtsa cells were maintained in DMEM media as previously described [12 (link)]. Primary human umbilical vein endothelial cell (HUVEC, Cambrex) was cultured in EBM-2 basal media supplemented with EGM-2 MV Kit (Lonza) containing 2% FBS. HUVEC cells of passage 6 to 8 were used. To ensure optimal siRNA delivery in xenograft tumors, in vivo-jetPEI (Polyplus-transfection Inc. NYC, NY), a linear polyethylenimine, was used in conjunction with siRNA [13 (link)].

Human umbilical vein endothelial cells (HUVEC) were purchased from Cambrex Bio Science Walkersville. These cells were cultured in Endothelial basal medium-2 (EBM-2, Lonza, Walkersville, MD, USA) supplemented with 2% fetal bovine serum (FBS, Lonza) and endothelial cell growth factors (Lonza, Hydrocortisone 0.2 ml, hFGF-B 2 ml, VEGF 0.5 ml, R3-IGF-1 0.5 ml, Ascorbic acid 0.5 ml, hEGF 0.5 ml, GA-1000 0.5 ml, Heparin 0.5 ml). Before cells were seeded, 18 mm round coverslips (Fisherbrand, Leicestershire, United Kingdom) were coated with 2% gelatin (Gelatin from porcine skin Type A, sigma). Before using the coverslip, it was dipped into 100% ethanol and flame sterilized. 500 μl of 2% gelatin solution in phosphate buffered saline (PBS) was added to 18 mm coverslips and incubated for 2 hours at 37°C. After 2 hours, 2% gelatin solution was suctioned and dried in air. For individual cell migration model (nonconfluent), 8 × 10³ cells in 600 μl were plated on the coverslip at 30% confluency. The cells were plated onto gelatin-coated coverslips and were incubated for 24 hours before exposure to flow. HUVEC were studied before passage 10 in all experiments.

Human umbilical cord vein endothelial cells (HUVEC) were from Cambrex and were maintained in basal Endothelial Growth Medium (EGM-2) and 10% fetal calf serum (FCS, Hyclone). Cells were split 1:3 twice a week, and used until they reached passage seven.

Human Umbilical cord vein endothelial cells (HUVEC) were from Cambrex and were maintained in basal EGM-2 and 10% FBS (Hyclone). Cells were split 1∶3 twice a week, and used until they reached passage 7. Primary human hematopoietic-proangiogenic cells (PACs, CD133+ and CD34+ cells) isolated from human bone marrow, were from Lonza (2M-102), and were maintained in IMDM and 15% FBS. Cells were split 1∶2 once a week, and used until they reached passage 5.

Human Umbilical Vein Endothelial Cells (HUVEC) purchased from Cambrex Corp. were cultured in complete EGM-2 medium (Lonza, Switzerland). Passage 3-10 HUVEC maintained in confluency for up to 3 days were used. K562 cells were grown in RPMI 1640 media containing 10% fetal bovine serum (FBS) and pen/strep. HeLa cells and Raw 264.7 cells were cultured in DMEM media containing 10% FBS and pen/strep. Anti-CBP mAb that was used in the ChIP assays recognizes both CBP and p300 was from ABCAM (Cambridge UK).