DMEM/F12 (Invitrogen) supplemented with 10% FBS (Hyclone Laboratory), except

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1 Materials and Methods Cell Culture Rat aortic SMCs were isolated as previously described 1 and then cultured in DMEM/F12 (Invitrogen) supplemented with 10% FBS (Hyclone Laboratory), except where otherwise indicated. Cells were used between passages 3 and 12. For palmitate treatment, confluent SMCs were cultured for 48 h in a defined serum-free DMEM/F12 supplemented with 0.68 mmol/l L-glutamine, 5x10-7 mol/l insulin, 2x10-4 mol/l L-ascorbic acid, and 5 g/ml transferrin, 8x10-5 mol/l selenium, and 100 units/ml penicillin (Life Technologies) and 100 µg/ml streptomycin (Life Technologies) 2, 3. The cells were then treated for 24 h with either 200 mol/l palmitate or vehicle (fatty acid-free bovine serum albumin [BSA]) (Sigma). Sodium palmitate (Sigma) was dissolved in warm 50% ethanol to make a 100 mmol/l palmitate stock solution 4. To make 5 mmol/l palmitate/10% fatty acid-free BSA solution, the palmitate stock solution and 10% fatty acid-free BSA in water were incubated for 10 min at 55ºC, mixed and incubated for an additional 10 min at 55ºC to obtain a homogeneous mixture.. To analyze the effects of inhibitors, SMCs were pretreated with 10 mmol/l N-acetyl-L-cysteine (NAC) (Sigma), 100 mo/l apocynin (Sigma), or 5 mol/l BMS (Sigma) for 1 h prior to the addition of palmitate. Quantitative Real-Time PCR Total RNA was extracted from cultured cells using RNeasy (Qiagen) and from arteries using an RNeasy fibrous kit (Qiagen). Real-time PCR was carried out using LightCycler

2 (Roche) and QuantiTect SYBR green PCR kits (Qiagen). The sequences of the PCR primers used are shown in Supplemental Table 1. Western Blotting Cytoplasmic and nuclear extracts were obtained using NE-PER nuclear and cytoplasmic extraction reagent (Thermo Scientific) with protease inhibitor and phosphatase inhibitors according to the supplier s protocol. Whole cell lysates were prepared in RIPA buffer containing 150 mmol/l NaCl, 50 mmol/l Tris-HCl (ph 7.5), 1% NP-40, 0.1% SDS, 0.5% Na-deoxycholate, 1 mmol/l DTT and Complete EDTA-free protease inhibitors (Roche). Antibodies against phospho-ikk and I B-α were purchased from Cell Signaling. Antibodies against NF- B p65, NOX1 and β-tubulin were from Santa Cruz. Animals Eight-week-old male C57BL/6 mice (body weight 20 to 25 g) were purchased from Japan Clea, and Myd88 -/- mice of the C57BL/6 background were from Oriental Bio. All mice were fed a normal rodent chow diet. Ligation of the right common carotid artery was performed as previously described 5. After ligation, the mice were intraperitoneally administered either 0.6 mg/g emulsified ethyl palmitate or vehicle daily for 3 weeks. Ethyl palmitate (Tokyo Chemical Industry) was dissolved with 1.6% lecithin (Wako) and 3.3% glycerol in water to produce a mixture containing 600 mmol/l ethyl palmitate, 1.2% lecithin and 2.5% glycerol 6. The mixture was then emulsified using sonicator. The lecithin-glycerol-water solution was used as the vehicle. To measure serum palmitate, a

3 single dose of either ethyl palmitate emulsion or vehicle was intraperitoneally administered to mice that had been fasted for 16 h, and serum was collected 6 h later. To measure FFA levels, mice fed a normal chow diet were intraperitoneally administered either ethyl palmitate or vehicle daily. For BM transplantation, BM cells were obtained from 8-week-old C57BL/6 (Ly5.1) mice and transplanted into lethally irradiated wild-type C57BL/6 and Myd88 -/- mice (Ly5.2). Flow cytometric analysis using Ly-5.1 antibody indicated that >85% of peripheral blood leukocytes were reconstituted in the BM-transplant recipient mice. Six weeks after the transplantation, the mice were subjected to the carotid artery ligation. FFA, Triglyceride and Cholesterol Measurements FFA, triglyceride and cholesterol levels were respectively measured using a NEFA C kit, L-type WAKO TG kit and Cholesterol E test WAKO (Wako), following the instructed protocols. Serum Palmitate Measurement Serum lipids were extracted and separated by thin-layer chromatography (TLC) using hexane/diethyl ether/acetic acid (80:20:1, v/v). Free fatty acid spots were visualized by iodine vapors, scraped and methylated with 1% H 2 SO 4 in methanol. Methyl palmitate was then quantified by GS-MS analysis.

4 Immunohistochemical Analysis Carotid arteries were fixed with 40% Ufix (Sakura Finetek) overnight and then immunohistologically stained using anti-sm α-actin (Sigma), anti-sm1 (KM3660, Kyowa Medex) and anti-mac3 (BD Biosciences) as primary antibodies. ROS Measurement To measure ROS, SMCs were treated with BSA or palmitate for 24 h, after which the cells were rinsed twice with PBS and incubated with 10 μmol/l CM-H 2 DCFDA (Invitrogen) for 30 min at 37ºC. ROS fluorescence was then detected using a flow cytometer (BD FACSCalibur). Lucigenin-enhanced chemiluminescence was also used to measure the level of superoxide in cells. After stimulation for 24 h with BSA or palmitate, SMCs were incubated with DETCA (3 mmol/l) in Krebs-HEPES buffer for 1 h to inactivate endogenous SOD activity. The medium was then exchanged with a fresh KRB buffer, and chemiluminescence was detected using lucigenin (5 mol/l) and NADPH (100 mol/l). The buffer blank background was subtracted from each reading, and the area under the curve during the 3 min immediately after the addition of lucigenin was calculated. Bromodeoxyuridine (BrdU) Incorporation SMC proliferation was examined using a BrdU cell proliferation assay kit (Roche) according to the manufacturer s protocol. SMCs were incubated for 4 h in medium containing BSA or 200 mol/l palmitate plus BrdU, after which the absorbance of the samples at 405 nm was measured.

5 sirna sirna was synthesized using a silencer sirna construction kit (Ambion) according to the manufacture s instructions. The template sequences were as follows: Nox1, CAGAGGCAAGAGATCCATC; Myd88, ACCACCATGCGACGACACC; and Tlr4, TCTCAATTGCTTGGCAAAT. A sirna against secreted alkaline phosphatase (SEAP) served as a control 7. For transfection, rat aortic SMCs were plated in 3.5-cm culture dishes. Once the cells reached 50% confluency, 40 pmol of sirna were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacture s protocol. The cells were incubated in growth medium without antibiotics (Invitrogen) containing the sirna-lipofectamine 2000 complex for 4 h, after which the medium was replaced with growth medium, and the incubation was continued until the cells reached confluence (20 h). The medium was then replaced with the serum-free defined medium 3, 8 and the incubation was continued for 24 h prior to palmitate treatment. Plasmids and Luciferase Assay The rat Nox1 promoter region was obtained by PCR. Promoter fragments were then subcloned into pgl3 basic luciferase vector. In addition, an NF- B binding site mutant construct, in which the potential B element at -264 bp (5 - TGATATTTCC-3 ) was mutated to 5 -TGACCATATG-3 was generated by PCR. In both cases, luciferase activity was normalized to the protein concentration of each cell lysate 9. For transfection, SMCs were plated to a density of 2.0x10 4 cells/cm 2 in 12-well plates, and the next day luciferase reporter constructs were transfected using Polyfect (Qiagen) according to the manufacture s instructions. After an additional 24 h, the cells were

6 cultured in growth medium supplemented with 10% FBS until they reached confluence. The medium was then replaced with serum-free defined medium for 24 h, followed by an additional 24 h with palmitate, after which they were subjected to luciferase assays. Chromatin Immunoprecipitation (ChIP) ChIP assays was performed as previously described 9. Briefly, 2x10 6 cells were grown at confluence for 24 h and then cultured in serum-free defined medium for an additional 24 h, after which either palmitate (200 mol/l) or vehicle was added to the medium for 8 h. The cells were then cross-linked by adding formaldehyde to a final concentration of 1%, and chromatin samples were prepared. Anti-NF- B p65 antibody was used for immunoprecipitation (Santa Cruz sc-372x). The primers used to detect the Nox1 promoter were 5 TGGAGTGCGTTGAGCCCAGCTG- 3 and 5 -CAAAGTCGGCTTAGAACTGGATC-3. Statistical Analysis Student t test was used for comparisons between two groups. Differences among more than two groups were analyzed using one-way ANOVA followed by a post hoc Tukey-Kramer test for multiple groups. Values of P<0.05 were considered significant. References 1. Shimizu RT, Blank RS, Jervis R, Lawrenz-Smith SC, Owens GK. The smooth muscle alpha-actin gene promoter is differentially regulated in smooth muscle versus non-smooth muscle cells. J Biol Chem. 1995;270:

7 2. Kihm AJ, Hershey JC, Haystead TA, Madsen CS, Owens GK. Phosphorylation of the rrna transcription factor upstream binding factor promotes its association with TATA binding protein. Proc Natl Acad Sci U S A. 1998;95: Everett AD, Lobe DR, Matsumura ME, Nakamura H, McNamara CA. Hepatoma-derived growth factor stimulates smooth muscle cell growth and is expressed in vascular development. The Journal of clinical investigation. 2000;105: Rho M-C, Ah Lee K, Mi Kim S, Sik Lee C, Jeong Jang M, Kook Kim Y, Sun Lee H, Hyun Choi Y, Yong Rhim B, Kim K. Sensitization of vascular smooth muscle cell to TNF-α-mediated death in the presence of palmitate. Toxicol Appl Pharmacol. 2007;220: Choi ET, Khan MF, Leidenfrost JE, Collins ET, Boc KP, Villa BR, Novack DV, Parks WC, Abendschein DR. Beta3-integrin mediates smooth muscle cell accumulation in neointima after carotid ligation in mice. Circulation. 2004;109: Eguchi K, Manabe I, Oishi-Tanaka Y, Ohsugi M, Kono N, Ogata F, Yagi N, Ohto U, Kimoto M, Miyake K, Tobe K, Arai H, Kadowaki T, Nagai R. Saturated Fatty Acid and TLR Signaling Link b Cell Dysfunction and Islet Inflammation. Cell Metab. 2012;15: Yagi N, Manabe I, Tottori T, Ishihara A, Ogata F, Kim JH, Nishimura S, Fujiu K, Oishi Y, Itaka K, Kato Y, Yamauchi M, Nagai R. A nanoparticle system specifically designed to deliver short interfering RNA inhibits tumor growth in vivo. Cancer Res. 2009;69:

8 8. Fujiu K, Manabe I, Ishihara A, Oishi Y, Iwata H, Nishimura G, Shindo T, Maemura K, Kagechika H, Shudo K, Nagai R. Synthetic retinoid Am80 suppresses smooth muscle phenotypic modulation and in-stent neointima formation by inhibiting KLF5. Circ Res. 2005;97: Manabe I, Owens GK. CArG elements control smooth muscle subtype specific expression of smooth muscle myosin in vivo. J Clin Invest. 2001;107: