Table 1.
Antibodies used in this study.
Figure 1.
Decreased lipid uptake and immunological signaling in human ALOX15B-silenced macrophages.
Lipid accumulation was analyzed in human primary macrophages transfected with nonsilencing control siRNA or ALOX15B siRNA using Oil Red O staining after incubation with DMOG. A) Quantification of ALOX15B expression normalized to ActB expression measured with Q-PCR. B) Quantification of ALOX15A expression normalized to ActB expression measured with Q-PCR. C) Representative picture showing Oil Red O staining of control and ALOX15B-silenced macrophages (Scale bar = 50 µm). D) Quantification of Oil Red O staining in human primary macrophages (n = 7) normalized to control. E) Size of control and ALOX15B-silenced human primary macrophages (foam cells). F) Quantification of secreted cytokines in media from human primary macrophages (n = 7). Data are presented as mean±SEM normalized to control.
Figure 2.
Alox15b knockdown in LDLr−/− mice.
A) Immunohistochemical detection of macrophages and ALOX15B in Ldlr−/− mice. B) Quantification of Alox15b expression, normalized to Emr1 expression (macrophage marker) in aortic tissue using Q-PCR (n = 2 for control and n = 3 for Alox15b shRNA). The sections presented in figure 2A were stained with Mayer's hematoxylin while the quantified sections used for figure 2B were not. C) Western blotting of ALOX15B and MAC-2 (macrophage marker) in aortic tissue. D) Quantification of Alox15b 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 Alox15b shRNA). E) Western blotting of ALOX15B and ACTB in bone marrow macrophages. F) ALOX15B levels measured by immunohistochemistry in sections from aortic sinus from control and Alox15b knockdown mice (n = 7 per group). Data are presented as mean±SEM.
Figure 3.
Decreased atherosclerotic lesions in aortas in Alox15b knockdown mice.
A) Plasma cholesterol, B) plasma triglycerides and C) body weight of Alox15b knockdown and control mice. D) Representative photographs showing aorta pinned out by en face technique and stained with Sudan IV. E) Quantification of subendothelial lipid accumulation in the aorta (n = 7 per group). F) Representative histological analysis of the aortic sinus stained with Oil Red O. G) Quantification of subendothelial lipid accumulation in the aortic root (n = 6 per group). Data are presented as mean±SEM.
Figure 4.
Analysis of plaque composition and plasma levels of IL-2.
Sections from aortic sinuses were stained with antibodies against A) CD4/CD8 (T cells), B) MAC-2 (macrophages), C) α-actin (smooth muscle cells), and D) collagen. Data are presented as mean±SEM. E) Plasma levels of soluble IL-2. (n = 6 per group).