Association of a polymorphism of BTN2A1 with myocardial infarction in East Asian populations Yoshiji Yamada, Tamotsu Nishida, Sahoko Ichihara, Motoji.

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Association of a polymorphism of BTN2A1 with myocardial infarction in East Asian populations Yoshiji Yamada, Tamotsu Nishida, Sahoko Ichihara, Motoji Sawabe, Noriyuki Fuku, Yutaka Nishigaki, Yukitoshi Aoyagi, Masashi Tanaka, Yoshinori Fujiwara, Hiroto Yoshida, Shoji Shinkai, Kei Satoh, Kimihiko Kato, Tetsuo Fujimaki, Kiyoshi Yokoi, Mitsutoshi Oguri, Tetsuro Yoshida, Sachiro Watanabe, Yoshinori Nozawa, Aki Hasegawa, Toshio Kojima, Bok-Ghee Han, Younjin Ahn, Meehee Lee, Dong-Jik Shin, Jong Ho Lee, Yangsoo Jang Atherosclerosis Volume 215, Issue 1, Pages 145-152 (March 2011) DOI: 10.1016/j.atherosclerosis.2010.12.005 Copyright © 2010 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 Effect of rs6929846 in BTN2A1 on transcriptional activity. HEK293T cells were transfected with a luciferase reporter gene linked to the C or T allele of rs6929846 or with the reporter gene alone (control), after which luciferase activity of cell lysates was measured. Relative luciferase activity (firefly luciferase activity/Renilla luciferase activity) was calculated, and that for the reporter gene alone (control) was arbitrarily set to 1.0. Data are means±SE from 10 independent experiments, with each experiment being performed in duplicate. Atherosclerosis 2011 215, 145-152DOI: (10.1016/j.atherosclerosis.2010.12.005) Copyright © 2010 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 Expression of BTN2A1 and atherosclerosis-related genes in HEK293T cells transfected with human BTN2A1 cDNA. (A) Immunoblot analysis of BTN2A1 in HEK293T cells transfected for 24h with pCMV6-AC containing human BTN2A1 cDNA or with the empty vector alone (control). β-Actin was examined as a loading control. Data are representative of three independent experiments. (B) Quantitative RT-PCR analysis of elastin (ELN), matrix metallopeptidase 3 (MMP3), and interleukin 5 (IL5) gene expression in HEK293T cells transfected as in (A). Data were normalized by the mean abundance of mRNAs for B2M, HPRT1, RPL13A, GAPDH, and ACTB, are expressed relative to the normalized value for control cells, and are means from two independent experiments. Atherosclerosis 2011 215, 145-152DOI: (10.1016/j.atherosclerosis.2010.12.005) Copyright © 2010 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 Immunohistochemical analysis of BTN2A1 and ILF3 in human coronary arteries. (A) A culprit MI lesion of a human coronary artery stained with hematoxylin–eosin. A hemorrhagic plaque with a necrotic core resulted in marked luminal stenosis. (B and D) Immunohistochemical staining for BTN2A1 revealed immunoreactivity in medial smooth muscle cells (SMC), cardiac muscle fibers, and endothelial cells (EC). The boxed region in (B) is shown at higher magnification in (D). (C, E, and F) Immunohistochemical staining for ILF3 revealed immunoreactivity in most nuclei of all cell types, including endothelial and smooth muscle cells, lymphocytes, macrophages, and subendothelial myofibroblasts (C and E). The acellular matrix of the necrotic core was also positive for ILF3 (C and F). Scale bars: 1mm in (A), (B), and (C); 50μm in (D), (E), and (F). Atherosclerosis 2011 215, 145-152DOI: (10.1016/j.atherosclerosis.2010.12.005) Copyright © 2010 Elsevier Ireland Ltd Terms and Conditions