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1 Supporting Information Horie et al /pnas SI Materials and Methods ell ulture and Reagents. THP-1 cells were obtained from the American Type ell ollection. THP-1 cells were transformed into macrophages by incubation for 3 d with 100 nm PMA (Nacalai Tesque). An immortalized primary human hepatocyte (HuS-E/2) cell line was kindly given by Makoto Hijikata (Kyoto University, Kyoto, Japan). Peritoneal macrophages were obtained from the peritoneal cavity of WT and mir-33 deficient mice 4 d after i.p. injection of 1 ml 10% thioglycolate. The cells obtained were washed with RPMI1640 (Nacalai Tesque), spun at 1,000 rpm for 10 min, and plated at a density of 10 6 cells per ml. ells were washed 1 h later and incubated for 2 d, then used for experiments. The antibodies used were a polyclonal anti-aa1 antibody (Novus iologicals), a polyclonal anti-ag1 antibody (Novus iologicals), a polyclonal anti SREP-2 antibody (ayman hemical), an anti-gaph antibody (ell Signaling Technology), and an anti β-actin antibody (Sigma-Aldrich). Simvastatin was purchased from Wako Pure hemical Industries. Acetylated LL (AcLL) was purchased from iomedical Technologies. ApoA-I was from Sigma-Aldrich. [1, 2-3 H (N)]-cholesterol was from Perkin-Elmer. Plasmids. Expression vectors for the negative control (mir-control) and micrornas were generated using LOK-iT PolIImiR RNAi Expression Vector Kits in accordance with the manufacturer s protocol (Invitrogen). The mir-control vector contains a hairpin structure just as for a regular premirna, but which is predicted not to target any known vertebrate gene (pcna6.2-gw/ EmGFP-miR-neg control plasmid). To create an anti mir-33 (decoy) vector, the luciferase 3 UTR was modified to include three to nine tandem sequences complementary to mir-33, separated by a single nucleotide space. The sequences of all constructs were analyzed using an AI 3100 genetic analyzer. All of these constructs were correctly inserted into a plenti6/v5--topo vector (Invitrogen) driven by a MV promoter to stably express genes in THP-1 and HuS-E/2 cells. Southern lotting. Southern blotting was performed using IG High Prime NA Labeling and etection Starter Kit II in accordance with the protocol of the manufacturer (Roche). Primer Sequences for the Southern lotting Probe and Genotyping. Primer sequences for the probe (865 bp) for Southern blotting and genotyping (WT, 385 bp; KO, 491 bp) were as follows: Southern probe primer sense; AATGAGTGAGAGGTG- GAGTTTG Southern probe primer antisense; ATGATTGAGTTA- GAGTA WT/KO sense; GGATATTTGATTT WT antisense; AATAAATGAAAGTG KO antisense; TTGGGATAGAATTGTGATTAA Western lotting. ell lysates were prepared and subjected to SS/ PAGE followed by standard Western blotting procedure. Quantitative PR for microrna. Measurement of mir-33 was performed in accordance with the TaqMan MicroRNA Assays (Applied iosystems) protocol, and products were analyzed using a thermal cycler (AI Prism7900HT sequence detection system). Values were normalized using U6 snrna expression. Quantitative PR for mrna. Total RNA was isolated using TRIzol reagent (Invitrogen). cna was synthesized using Transriptor First Strand cna Synthesis Kit (Roche), and PR was performed with a SYR Green PR master mix (Applied iosystems), normalized with GAPH. An AI Prism 7900HT sequence detection system was used as a thermal cycler. Genespecific primers were as follows: AA1 sense (human); 5 GTTTTTGATTATT- GG3 AA1 antisense (human); 5 AGTTTGGAAGGTTTG- TTA3 SREP2 sense (human); 5 AGGAGAAATGGTGTGA3 SREP2 antisense (human); 5 TAAAGGAGAGGAAG- GA3 LL-receptor sense (human); 5 AGATATATAAGAA- G3 LL-receptor antisense (human); 5 TTAAAGTT- AT3 GAPH sense (human and mouse); 5 TTGATAAGA- TT3 GAPH antisense (human and mouse); 5 TTGTATGGAT- GATTGG3 sense (mouse); 5 GTGGAGAGTTAAGTA3 antisense (mouse); 5 TGGTAGGTTAAG- GAG3 Oligonucleotide Sequences Used for onstruction of WT or Mutant Abca1 and Abcg1 3 UTR Reporter onstructs. WT Abca1: GAAAAATGGATATGTATGAATATTAATG- AATGATTAATGAAGAGAAAAATTAT- TA Mutant Abca1: GAAAAATGGATATGTATGAATATTATA- GTTAGTTTATAGTAGAGAAAAATTAT- TA WT Abcg1: TAGTAAAGTGGTGGGGAGAGGGAT- AAGAAGAATGAAGAATGAGAAGTG- TGGGGTATTA Mutant Abcg1: TAGTAAAGTGGTGGGGAGAGGGA- TAAGAAGATAGTAGATAGTGAAGTG- TGGGGTATTA 1of5

2 PMA(-) PMA(+) Phase Phase GFP GFP Fig. S1. Microscopy images of THP-1 and HuS-E/2 cells. (A) mirnas were transduced into THP-1 cells using lentivirus vectors. Transduction efficiency, which was shown using GFP, was always greater than 90%. THP-1 cells were induced to differentiate into macrophages by PMA stimulation (100 nm) for 3 d. () mirnas were transduced into HuS-E/2 cells, which are immortalized human primary hepatocytes. Transduction efficiency, which was shown using GFP, was always greater than 90%. A E A A 1 GA P H A A 1 Si mv ast at in 24 h ( µm) Si mv ast at in (10 µm) 0h 6h 24 h LL- r ecep to r/ GAP H SREP2 /G APH mi R- 33/ U6 GA P H 0h 6h 24 h 0h 6h 24 h 0h 6h 24 h Fig. S2. Effect of simvastatin in THP-1 macrophages. (A) Western blot analysis of AA1 protein levels after stimulation with simvastatin for 24 h at indicated concentrations in THP-1 macrophages. GAPH was used as loading control. () Western blot analysis of AA1 protein levels after stimulation with simvastatin (10 μm) for indicated time periods in THP-1 macrophages. GAPH was used as a loading control. () Quantitative real-time PR analysis of LL receptor expression levels following stimulation with simvastatin (10 μm) for indicated time periods in THP-1 macrophages. Values are mean ± SE of six independent experiments with normalization using Gapdh expression. P < 5 compared with 0 h. () Quantitative real-time PR analysis of expression levels following stimulation with simvastatin (10 μm) in THP-1 macrophages. Values are mean ± SE of six independent experiments with normalization using GAPH expression. P < 5 compared with 0 h. (E) Quantitative real-time PR analysis of mir-33 expression levels following stimulation with simvastatin (10 μm) for time indicated in THP-1 macrophages. Values are mean ± SE of six independent experiments with normalization using U6 snrna expression. P < 5 compared with 0 h. 2of5

3 MV ecoy(anti-mir-33)-luc x3 MV Anti-miR-33 Anti-miR-33 Anti-miR-33 x6 MV Anti-miR-33 Anti-miR-33 Anti-miR-33 x9 MV Anti-miR-33 Anti-miR-33 Anti-miR-33 Anti-miR-33 Tandem sequences complementary to mir-33 ecoy-lucx3 ecoy-lucx6 activity (%) AA1 ecoy(anti-mir-33x9)-luc ecoy(anti-mir-33x9)-luc ecoy(anti-mir-33x9)-luc ecoy-lucx9 GAPH 10% FS SFM Simvastatin (10 µm) Fig. S3. Silencing of endogenous mir-33 using decoy gene in vitro. (A) Schema of decoy gene. 3 UTR was modified to include three to nine tandem sequences complementary to mir-33, each separated by a single nucleotide spacer. () 293T cells were transfected with control-luc or decoy-luc ( 3, 6, and 9) constructs, along with expression vector of mir-33. activity was measured 48 h after transfection. Reduction in luciferase activity indicates effect of decoy gene. Values are mean ± SE of three independent experiments.p < 01; P < 5. () Western blot analysis of AA1. THP-1 cells were transfected with control-luc or decoy (anti mir-33 9)-luc using a lentivirus vector. ells were cultured in RPMI 1640 with 10% FS; otherwise cells were cultured without FS or treated with simvastatin (10 μm) for 24 h. GAPH was used as loading control. Re la ti ve expr essi on leve l Sr e bp2 (8 w eek) Ma le (+ /+ ) Ma le (-/ -) Fe ma le (+ /+ ) Fe ma le (-/ -) Fig. S4. omparison of expression in 8-wk-old mice. Quantitative real-time PR analysis of in liver of 8-wk-old male and female mice. Values are mean ± SE of three to four mice with normalization using Gapdh expression. Value for WT male mice was set at. 3of5

4 (exon16 exon17) Gapdh +/+ -/- +/+ -/ (16 week) Male (+/+) Male (-/-) Female (+/+) Female (-/-) (exon16 exon17) Gapdh +/+ -/- +/+ -/ (24 week) Male (+/+) Male (-/-) Female (+/+) Female (-/-) Fig. S5. omparison of expression in 16- and 24-wk-old mice. (A) RT-PR analysis of in the liver of 16-wk-old male (Upper) and female (Lower) mice. The sense primer was designed in exon 16 of and the antisense primer was designed in exon 17 of. Gapdh expression was used as a control. () Quantitative real-time PR analysis of in the liver of 16-wk-old male and female mice. Values are the means ± SE of three to four mice with normalization using Gapdh expression. The value for WT male mice was set at. () RT-PR analysis of in the liver of 24-wk-old male (Upper) and female (Lower) mice. Sense primer was designed in exon 16 of, and antisense primer was designed in exon 17 of. Gapdh expression was used as control. () Quantitative real-time PR analysis of in the liver of 24-wk-old male and female mice. Values are mean ± SE of three to four mice with normalization using Gapdh expression. Value for WT male mice was set at. 4of5

5 Re la ti ve Expr essi on leve l Re la ti ve ex pr essi on leve l Ep if at mi R- 33 Me sf at Su bf at Lu ng Sp l een Te st is He ar t Li ver Sm al li nt est in e Ki dne y Mu scl e r ai n mir-33 (Ki dney) +/ + -/ - +/ - Re la ti ve ex pr essi on leve l mir-33 ( ra in ) +/ + -/ - Re la ti ve ex pr essi on level Ep if at +/ - Me sf at Su bf at Lun g Spl een T est is H ear t Li ver Sm al li nt est in e Ki dne y Mu scl e r ai n Ma le (+ /+ ) mir-33 (Li ver ) Fig. S6. Expression level of mir-33 in mice. (A) Quantitative real-time PR analysis of mir-33 and in 8-wk-old WT male mice with normalization using U6 snrna or Gapdh expression. Values for liver were set at. () Quantitative real-time PR analysis of mir-33 in kidney of 8-wk-old male mice. N, not determined. () Quantitative real-time PR analysis of mir-33 in brain of 8-wk-old male mice. N, not determined. () Quantitative real-time PR analsysis of mir-33 in liver of 16-wk-old male and female mice. N, not determined. Ma le (-/ -) Fe ma le (+ /+ ) Fe ma le (-/ -) A Abcg1 3 UTR mir-33 binding site 1 2 Mmu (Mouse) Rno (Rat) UGGUGGGGAGAGGGAUAAGAAGA AUGA AG-----AA UGA GAAGUGUGGGG UGGUGGGAGAGGGAU A AUGA AUGAAAAA UGA GAAGUGUGGGG Relative luciferase activity Abcg1 3'UTR AG mir-control mir-33 mir-146a mir-control mir-33 mir-146a GAPH AG1 GAPH +/+ -/- +/+ + -/- WT Mutant Fig. S7. Analysis of AG1 in vitro and in vivo. (A) Sequence alignment of Abcg1 3 UTR. There are two potential mir-33 binding sites in the Abcg1 3 UTR; however, these were conserved only in rodents and not in humans. () 293T cells were transfected with WT or mutant Abcg1 3 UTR luciferase constructs, along with expression plasmids for mir-control (negative control), mir-33, and mir-146a. Values are mean ± SE of four independent experiments. P < 5 compared with other columns. () Western blot analysis of hepatic AG1 in 16-wk-old male mice. GAPH was used as loading control. () Western blot analysis of hepatic AG1 in 16-wk-old female mice. GAPH was used as loading control. 5of5

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