Fig 2: Effect of cell density on E-selectin protein expression levels in HUVECs.(A) HUVECs were cultured as described in the Materials and Methods. Photomicrographs were obtained using phase-contrast microscopy (20× magnification). (B) E-selectin protein expression levels under the sparse (S) and confluent (C) conditions. HUVECs cultured under both the conditions were treated with (+) or without (-) 1 ng/ml TNFa for 4 h, and total cell extracts were prepared. Equal amounts of protein were separated using SDS-PAGE, and western blot analysis was performed using the anti-E-selectin antibody (Santa Cruz Biotechnology). (C) Flow cytometric analysis of E-selectin cell surface expression. HUVECs were stimulated with or without 1 ng/ml TNFa for 4 h, and flow cytometry was performed using the anti-E-selectin antibody (clone 7A9) (*p<0.05). The values are expressed as mean ± SD of three independent experiments.

Fig 3: Chromatin accessibility differed between sparse and confluent HUVEC monolayers.CHART-PCR of the E-selectin promoter region. The location of primers used in CHART-PCR assays is shown. Nuclei were isolated from sparse and confluent HUVECs monolayers treated with (+) or without (-) 1 ng/ml TNFa for 60 min. The isolated nuclei were incubated with 10 U of micrococcal nuclease for 10 min at room temperature. Genomic DNA was extracted and quantified using real-time PCR relative to DNA prepared from undigested nuclei (*p<0.05, **p<0.01). The values are expressed as mean ± SD of three independent experiments. CRR indicates cytokine response region.

Fig 4: Effect of cell density on histone H3 acetylation at the E-selectin promoter.Schema of the E-selectin gene and the location of the primers used in the ChIP assay. Chromatin was prepared from sparse and confluent HUVECs monolayers treated with (+) or without (−) 1 ng/ml TNFα for 30 min, and the ChIP assay was performed using the anti-acetyl histone H3 (AcH3) antibody. Real-time PCR analysis of the ChIP samples was performed using primers designed to amplify the E-selectin promoter region (−195 to −67), which contains the cytokine response region (CRR) and the E-selectin extreme 3′ region (+13914 to +13977) (*p<0.01). The values are expressed as mean ± SD of three independent experiments.