Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza Walkersville, Inc. (Walkersville, MD, USA) and cultured in endothelial basal medium (EBM)-2 complete medium supplemented with 2% (v/v) heat-inactivated fetal bovine serum at 37°C in 5% CO2. To examine the effects of paricalcitol on the LPS-induced increase in cell adhesion molecule expression in HUVECs, subconfluent endothelial cells were incubated with paricalcitol (Tocris Bioscience, Minneapolis, MN, USA; 0.01, 0.1 or 1 µM) for 30 min and then stimulated with 10 ng/ml TNF-α (R&D Systems, Minneapolis, MN, USA) for 6 h.
For p65 immunocytochemistry, the HUVECs were cultured in 6-well plates and treated with 1 µM paricalcitol for 30 min and then stimulated with 10 ng/ml TNF-α for 6 h. Following 4% paraformaldehyde fixation, the cells were washed with phosphate-buffered saline (PBS), permeabilized in 0.25% Triton X-100 and blocked with 1% goat serum. The HUVECs were then incubated with primary anti-p65 antibody (#4764; Cell Signaling Technology, Beverly, MA, USA), followed by staining with Cy3-conjugated secondary antibody (AP182C; Chemicon, Temecula, CA, USA). For nuclear staining, the HUVECs were incubated with 4′,6-diamidino-2-phenylinole (DAPI; Molecular Probes, Eugene, OR, USA).
For p65 immunocytochemistry, the HUVECs were cultured in 6-well plates and treated with 1 µM paricalcitol for 30 min and then stimulated with 10 ng/ml TNF-α for 6 h. Following 4% paraformaldehyde fixation, the cells were washed with phosphate-buffered saline (PBS), permeabilized in 0.25% Triton X-100 and blocked with 1% goat serum. The HUVECs were then incubated with primary anti-p65 antibody (#4764; Cell Signaling Technology, Beverly, MA, USA), followed by staining with Cy3-conjugated secondary antibody (AP182C; Chemicon, Temecula, CA, USA). For nuclear staining, the HUVECs were incubated with 4′,6-diamidino-2-phenylinole (DAPI; Molecular Probes, Eugene, OR, USA).