Effect of cell density on histone H3 acetylation at the E-selectin promoter.

<p>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) (*<i>p</i><0.01). The values are expressed as mean ± SD of three independent experiments.</p

Effect of cell density on TNFα-induced MAPK and NF-kB activation in HUVECs.

<p>Western blot of phosphorylated (p) and total JNK, p38 MAPK (<b>A</b>), and NF-κB p65 (<b>B</b>). HUVECs were stimulated with (+) or without (−) 1 ng/ml TNFα for 15 min. (<b>C</b>) p65 was detected using western blot analysis of the nuclear and cytoplasmic fractions prepared from HUVECs treated with or without 1ng/ml TNFα for 30 min. Representative western blots are shown.</p

Chromatin accessibility differed between sparse and confluent HUVEC monolayers.

<p>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 TNFα 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 (*<i>p</i><0.05, **<i>p</i><0.01). The values are expressed as mean ± SD of three independent experiments. CRR indicates cytokine response region.</p

Effect of cell density on E-selectin mRNA expression and mRNA stability in HUVECs.

<p>(<b>A</b>) Relative E-selectin mRNA levels measured using quantitative RT-PCR. RNA was isolated 2 h after stimulation with 1 ng/ml TNFα and 5 ng cDNA was used in each reaction (*<i>p</i><0.05 vs sparse). (<b>B</b>) E-selectin mRNA stability in TNFα-activated HUVECs. HUVECs were stimulated with 1 ng/ml TNFα for 2 h and treated with actinomycine D (5 µg/ml) for the indicated time periods. RNA was subjected to real-time RT-PCR. For each condition, E-selectin expression levels were normalized against those of GAPDH. The values were expressed proportional to that at baseline (time zero). The values are expressed as mean ± SD of three independent experiments.</p

Effect of cell density on E-selectin protein expression levels in HUVECs.

<p>(<b>A</b>) HUVECs were cultured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090502#s2" target="_blank">Materials and Methods</a>. Photomicrographs were obtained using phase-contrast microscopy (20× magnification). (<b>B</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 TNFα 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). (<b>C</b>) Flow cytometric analysis of E-selectin cell surface expression. HUVECs were stimulated with or without 1 ng/ml TNFα for 4 h, and flow cytometry was performed using the anti-E-selectin antibody (clone 7A9) (*<i>p</i><0.05). The values are expressed as mean ± SD of three independent experiments.</p

Cholesterol levels in lipoproteins and triglyceride levels.

<p>Cholesterol levels in lipoproteins and triglyceride levels.</p

Cholesterol proportions in lipoprotein fractions in Stage 4 group compared with those in Stage 5 group.

<p>Cholesterol proportions in lipoprotein fractions in Stage 4 group compared with those in Stage 5 group.</p

FGF23 level and cholesterol proportions and lipoprotein particle numbers in lipoprotein fractions.

<p>FGF23 level and cholesterol proportions and lipoprotein particle numbers in lipoprotein fractions.</p

Clinical and biochemical characteristics of patients in this study.

<p>Clinical and biochemical characteristics of patients in this study.</p

ABI and cholesterol proportions and lipoprotein particle numbers in lipoprotein fractions.

<p>ABI and cholesterol proportions and lipoprotein particle numbers in lipoprotein fractions.</p