<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

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