Lipid classes in human tissue sections from normal kidney tissue and clear-cell RCC tissue (CCRCC).

<p>All values are nmol/mg. Data are mean ± SEM.</p

VLDL-R overexpression in clear-cell RCC cells is mediated by HIF-1α, and promotes increased lipid accumulation through increased lipid uptake.

<p>(<b>A</b>) Quantification of VLDL-R mRNA normalized to 18S mRNA from cultured human cells isolated from healthy kidney tissue and clear-cell RCC tissue treated with siRNA against HIF-1α or VLDL-R (<i>n</i> = 10, *<i>p</i>≤0.05 vs. control siRNA normal cells, †<i>p</i>≤0.05 vs. control siRNA clear-cell RCC cells). (<b>B</b>) Quantification of immunoblot against VLDL-R with β-actin as loading control from cultured human cells isolated from healthy kidney tissue and clear-cell RCC tissue treated with siRNA against HIF-1α or VLDL-R (<i>n</i> = 10, *<i>p</i>≤0.05 vs. control siRNA normal cells, †<i>p</i>≤0.05 vs. control siRNA clear-cell RCC cells). (<b>C</b>) Quantification of Oil Red O staining of cultured human cells isolated from healthy kidney tissue and clear-cell RCC tissue treated with siRNA against HIF-1α or VLDL-R (<i>n</i> = 10, *<i>p</i>≤0.001 vs. control siRNA normal cells, †<i>p</i>≤0.05 vs. control siRNA clear-cell RCC cells). (<b>D</b>) Quantification of fluorescently internalized DiI-labeled lipoproteins in cultured human cells isolated from healthy kidney tissue and clear-cell RCC tissue treated with siRNA against HIF-1α or VLDL-R (<i>n</i> = 5, *<i>p</i>≤0.05 vs. control siRNA normal cells, †<i>p</i>≤0.05 vs. control siRNA clear-cell RCC cells). Data are shown as mean ± SEM.</p

VLDL-R expression is increased in clear-cell RCC.

<p>(<b>A</b>) Oil Red O staining of human tissue sections from normal kidney tissue (upper) and clear-cell RCC tissue (CCRCC; lower). (<b>B</b>) VLDL-R immunostaining (left) and quantification of immunostaining (right) of human tissue sections from normal kidney tissue (upper) and clear-cell RCC tissue (lower) (<i>n</i> = 6, *<i>p</i> = 0.0022). (<b>C</b>) Oil Red O staining (left) and quantification of staining (right) of cultured human cells isolated from healthy kidney tissue (upper) and clear-cell RCC tissue (lower) (<i>n</i> = 10, *<i>p</i> = 0.0058). (<b>D</b>) Quantification of VLDL-R mRNA normalized to 18S mRNA from cultured human cells isolated from healthy kidney tissue and clear-cell RCC tissue (<i>n</i> = 6, *<i>p</i> = 0.004). (<b>E</b>) Quantification of immunoblot against VLDL-R with β-actin as loading control from cultured human cells isolated from healthy kidney tissue and clear-cell RCC tissue (<i>n</i> = 6, *<i>p</i> = 0.0002). Data are shown as mean ± SEM.</p

<i>Alox15b</i> knockdown in <i>LDLr^−/−</i> mice.

<p><b>A</b>) Immunohistochemical detection of macrophages and ALOX15B in <i>Ldlr^−/−</i> mice. <b>B</b>) Quantification of <i>Alox15b</i> expression, normalized to <i>Emr1</i> expression (macrophage marker) in aortic tissue using Q-PCR (n = 2 for control and n = 3 for <i>Alox15b</i> shRNA). The sections presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043142#pone-0043142-g002" target="_blank">figure 2A</a> were stained with Mayer's hematoxylin while the quantified sections used for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043142#pone-0043142-g002" target="_blank">figure 2B</a> were not. <b>C</b>) Western blotting of ALOX15B and MAC-2 (macrophage marker) in aortic tissue. <b>D</b>) Quantification of <i>Alox15b</i> expression in bone marrow macrophages (BMM) isolated and differentiated at the end of the silencing experiment using Q-PCR (n = 2 for control and n = 3 for <i>Alox15b</i> shRNA). <b>E</b>) Western blotting of ALOX15B and ACTB in bone marrow macrophages. <b>F</b>) ALOX15B levels measured by immunohistochemistry in sections from aortic sinus from control and <i>Alox15b</i> knockdown mice (n = 7 per group). Data are presented as mean±SEM.</p

Analysis of plaque composition and plasma levels of IL-2.

<p>Sections from aortic sinuses were stained with antibodies against <b>A</b>) CD4/CD8 (T cells), <b>B</b>) MAC-2 (macrophages), <b>C</b>) α-actin (smooth muscle cells), and <b>D</b>) collagen. Data are presented as mean±SEM. <b>E</b>) Plasma levels of soluble IL-2. (n = 6 per group).</p

Decreased lipid uptake and immunological signaling in human <i>ALOX15B</i>-silenced macrophages.

<p>Lipid accumulation was analyzed in human primary macrophages transfected with nonsilencing control siRNA or <i>ALOX15B</i> siRNA using Oil Red O staining after incubation with DMOG. <b>A</b>) Quantification of <i>ALOX15B</i> expression normalized to <i>ActB</i> expression measured with Q-PCR. <b>B</b>) Quantification of <i>ALOX15A</i> expression normalized to <i>ActB</i> expression measured with Q-PCR. <b>C</b>) Representative picture showing Oil Red O staining of control and <i>ALOX15B</i>-silenced macrophages (Scale bar = 50 µm). <b>D</b>) Quantification of Oil Red O staining in human primary macrophages (n = 7) normalized to control. <b>E</b>) Size of control and <i>ALOX15B</i>-silenced human primary macrophages (foam cells). <b>F</b>) Quantification of secreted cytokines in media from human primary macrophages (n = 7). Data are presented as mean±SEM normalized to control.</p

Patient characteristics (n = 6).

<p>Patient characteristics (n = 6).</p

Severe depletion of ATP and glucose in perinecrotic zone of advanced plaques.

<p>A. Advanced atherosclerotic plaque, delineation in white of viable intima. Extract shows luminal (left) and perinecrotic zone (right) of viable intima. Note high expression of hexokinase II (HKII), indicative of hypoxia, in perinecrotic zone. B and C. Lower concentrations of ATP (B) and glucose (C) in perinecrotic zone (p<0.05). D and E. No significant difference in glycogen (D) and lactate concentrations (E) between luminal and perinecrotic zone. n = 6, paired t-test.</p

Antibodies used in this study.

1<p>Oxford Biochemical research,</p>2<p>BD Bioscience,</p>3<p>Nordic biosite,</p>4<p>Polyclonal,</p>5<p>Monoclonal,</p>6<p>Horse rabbit peroxidase,</p>7<p>GE healthcare.</p

Decreased atherosclerotic lesions in aortas in <i>Alox15b</i> knockdown mice.

<p><b>A</b>) Plasma cholesterol, <b>B</b>) plasma triglycerides and <b>C</b>) body weight of <i>Alox15b</i> knockdown and control mice. <b>D</b>) Representative photographs showing aorta pinned out by en face technique and stained with Sudan IV. <b>E</b>) Quantification of subendothelial lipid accumulation in the aorta (n = 7 per group). <b>F</b>) Representative histological analysis of the aortic sinus stained with Oil Red O. <b>G</b>) Quantification of subendothelial lipid accumulation in the aortic root (n = 6 per group). Data are presented as mean±SEM.</p