Huvecs

HUVECs (Lonza Group, Ltd.) were cultured in extracellular matrix (ECM; ScienCell Research Laboratories, Inc.) supplemented with 5% fetal bovine serum (FBS) and endothelial cell growth supplements (ScienCell Research Laboratories, Inc.); HUVECs of ≤6 passages were used. Human CRC cell lines SW480, SW620, SW48, LoVo and RKO were obtained from the American Type Culture Collection and cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS and 1% penicillin-streptomycin (P/S; Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. HUVECs were treated with 10 ng/ml recombinant human VEGF₁₆₅ (PeproTech, Inc.) for 3 or 6 h at 37°C. For the inhibition of VEGF signaling, HUVECs were treated with 5 µM VEGFR2 inhibitor DMH4 for 18 h at 37°C (Tocris Bioscience). For the western blot assays, HUVECs cultured for 20 h in either LoVo or SW620 CM as described below. At 20 h, harvested HUVECs were washed with PBS twice and lysed with RIPA lysis buffer (EMD Millipore) for quantification and blotting.

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).