NF-κB suppresses the expression of ATP-binding cassette transporter A1/G1 by regulating SREBP-2 and miR-33a in mice  Guo-Jun Zhao, Shi-Lin Tang, Yun-Cheng Lv, Xin-Ping Ouyang, Ping-Ping He, Feng Yao, Yan-Yan Tang, Min Zhang, Ya-Ling Tang, Deng-Pei Tang, Francisco S. Cayabyab, Guo-Ping Tian, Chao-Ke Tang  International Journal of Cardiology  Volume 171, Issue 3, Pages e93-e95 (February 2014) DOI: 10.1016/j.ijcard.2013.11.093 Copyright © 2013 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 NF-κB activation down-regulates ABCA1 and ABCG1 expression and up-regulates SREBP-2 and miR-33a expression in vitro. (A–C) RAW 264.7 macrophage derived foam cells were stimulated with LPS (10ng/ml) for 24h with or without PDTC (50μM). ABCA1, ABCG1, SREBP-2 and miR-33a mRNA expression levels were measured by RT-PCR. (D) A schematic diagram of the SREBP-2 promoter serial deletion reporter constructs and their potential NF-κB binding regions. (E) RAW264.7 macrophages were transfected with various reporter constructs (0.8μg) and pRL-TK vector (0.02μg) or mutated κBRE construct (0.8μg) and pRL-TK vector (0.02μg). Cells were starved overnight and treated with LPS (10ng/ml) with or without PDTC (50μM) for 24h. The reporter activities are shown as the relative luciferase units (RLU) normalized to the pRL-TK vector activity. Letters refer to diagrams in (D). (F) Cells were transfected with SREBP siRNA and subsequently incubated with or without LPS (10ng/ml) for 24h (PBS with no LPS acted as negative control). ABCA1 and ABCG1 protein levels were measured by Western blot. (G and H) ABCA1 and ABCG1 mRNA levels were measured by RT-PCR. The data represent means±SD from three separate experiments. *, P<0.05 vs. control. #, P<0.05 vs. LPS group. International Journal of Cardiology 2014 171, e93-e95DOI: (10.1016/j.ijcard.2013.11.093) Copyright © 2013 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 NF-κB activation down-regulates ABCA1 and ABCG1 expression and up-regulates SREBP-2 and miR-33a expression in vivo. Eight-week-old male apoE−/− mice were injected intraperitoneally with PBS (control), LPS (2.5mg/kg body wt) or LPS (2.5mg/kg body wt) plus PDTC (50mg/kg body wt) once a week for 8weeks. Four days prior to animal euthanasia, mice (n=15 per group) were intraperitoneally injected with thioglycollate to obtain thioglycollate-induced mouse peritoneal macrophages (MPMs). (A) Total protein extracts from cells were subjected to Western blot analyses for ABCA1 and ABCG1, while nuclear extracts were immunoblotted with SREBP-2 and NF-κB p65 antibody. β-Actin was used as normalization control. (B) Expression of miR-33a was confirmed by RT-PCR. (C) Representative Oil-red-O staining of aortic sinus lesion and quantification of the aortic sinus lesion area. Original magnification: ×40. All of the data represent the mean±SD from three separate experiments with triplicate samples. *, P<0.05 vs. control. #, P<0.05 vs. LPS group. International Journal of Cardiology 2014 171, e93-e95DOI: (10.1016/j.ijcard.2013.11.093) Copyright © 2013 Elsevier Ireland Ltd Terms and Conditions