Smooth Muscle Cells in Atherosclerosis Originate - Arteriosclerosis ...

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Fluorescence in situ hybridization (FISH) The Y chromosome was visualized in sections by a FITC-conjugated chromosome paint probe (Cambio, Cambridge, UK). Sections were first immunostained for SMαA, and high-power fields of SMαA + cells were photographed. Then, the cover slips were removed and the sections were pretreated in 10 mM sodium citrate, pH 6.0 (2h, 80°C) (Bie & Berntsen, Aarhus, Denmark), digested in pepsin solution (0.025%, 10 min, 37 °C, Sigma), fixed in 4% paraformaldehyde and treated in freshly prepared acetic acid (0.25% acetic anhydride in 0.1 M triethanolamine-HCl, pH 8.0, 10 min, room temperature (Sigma)) to reduce unspecific binding of the probe. Two μl paint probe was applied to dehydrated sections under cover slips, and probe and tissue were denatured at 60 °C for 10 min followed by overnight hybridization at 37 °C. Slides were washed in 50% formamide/1xSSC (15 min, 37°C) and in 2xSSC (15 min, 37°C), before incubation in DAPI solution and mounting in Slowfade Light Antifade. Supplementary References (1) Piedrahita JA, Zhang SH, Hagaman JR, Oliver PM, Maeda N. Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells. Proc Natl Acad Sci U S A. 1992 May 15;89:4471-4475. (2) Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y. 'Green mice' as a source of ubiquitous green cells. FEBS Lett. 1997 May 5;407:313-319. (3) Sata M, Saiura A, Kunisato A, Tojo A, Okada S, Tokuhisa T, Hirai H, Makuuchi M, Hirata Y, Nagai R. Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med. 2002;8:403-409. Downloaded from http://atvb.ahajournals.org/ by guest on June 4, 2013
Supplementary Table I. Plasma lipids in bone marrow transplanted mice at sacrifice Total cholesterol HDL cholesterol Triglycerides mmol/L mmol/L mmol/L 20 weeks of age (n=11) * 20.3±6.6 0.43±0.26 0.81±0.26 32 weeks of age (n=12) † *On high fat diet. † On chow diet. 11.7±1.6 0.31±0.18 0.79±0.31 Downloaded from http://atvb.ahajournals.org/ by guest on June 4, 2013
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Page 3 and 4: BM as performed in that experiment
Page 5 and 6: plaques from the control apoE / and
Page 7 and 8: The finding of intimal SMCs that do
Page 9 and 10: Supplementary Methods Transgenic mi
Page 11: and immersion-fixed for 6 hours at
Page 15: Supplementary Figure II Supplementa

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