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1 DOI: /ncb2210 b. ICAM1 ng ml -1 P = Small RNA (15-30nts) ng ml -1 Cell Lysate Exosome HDL Plasma HDL Normal Human HDL mirnas R = 0.45 P < Normal Human Exosome mirnas Figure S1. Characterization of HDL and exosome mirna profiles. ) Quantification of intercellular adhesion molecule 1 (ICAM1) protein ng ml -1 by ELISA. Huh7 cell lysate (n=4); exosome (n=4); and HDL (n=4). ICAM1 is an exosome protein marker. Data are means +/- s.e.m. b.) RiboQuant fluorescent quantification of gel-extracted small RNA (15% TBE-Urea gel, excised size ~15-30nt) ng ml -1. Human plasma (n=2) and human HDL (n=4). ) Spearman non-parametric correlation between human HDL and exosome mirna profiles from 3 matched subjects. Correlation of mirna mean relative quantitative values (RQV). 1

2 Antibody-Au b. Figure S2. Characterization of HDL-miRNA binding capacity. ) Transmission electron microscopy image of monoamino Nanogold-labeled antibody (1.4 nm). Control antibody immobilized on grid and goldenhanced for 2 min. Scale bar = 100nm. b.) Dynamic light scatter sizing of reconstituted HDL, rhdl. d = diameter. Data reported as size distribution by volume (%). 2

3 b. -Log10(p-value) Apoe-/- HFD v. WT Log2(Fold Change) -Log10(p-value) Apoe-/- Chow v. WT d. Log2(Fold Change) Apoe-/- HFD Apoe-/- Chow WT -Log10(p-value) Vickers Figure S3 Apoe-/- HFD v. Apoe-/- Chow Log2(Fold Change) Figure S3. Characterization of hyperlipidemia and atherosclerosis induced changes to mouse HDL-miRNA profiles. Human reconstituted HDL (rhdl), injected into wild-type (WT) (n=3), Apolipoprotein E null (Apoe-/-) chow diet (n=3), and Apoe-/- HFD (n=4) mice for 6h, retrieved distinct HDL-miRNA profiles. ) Hierarchal cluster analysis (heatmap) of mouse mirnas associated with injected rhdl from models of hyperlipidemia and atherosclerosis. Blue to red, color range gradient of mean abundance (-3 to 3). b.) Volcano plot of differentially abundant mouse mirnas associated with rhdl isolated from Apoe-/- HFD plasma compared to WT rhdl. Red dots, >1.5-fold change (Log2); P<0.1 (-Log10). ) Volcano plot of differentially abundant mouse mirnas associated with rhdl isolated from Apoe-/- chow plasma compared to WT rhdl. Blue dots, >1.5-fold change (Log2); P<0.1 (-Log10). d.) Volcano plot of differentially abundant mouse mirnas associated with rhdl isolated from Apoe-/- HFD plasma compared to Apoe-/- chow rhdl. Purple dots, >1.5-fold change (Log2); P<0.1 (-Log10). 3

4 RhoB mrna (percent control) Mock P = 0.02 mirna Nonsense mir-223 NPM1 b. M r (k) 62- GAPDH Cell Lysate Plasma HDL DGUC HDL FPLC HDL IP Exosome LDL Figure S4. Characterization of mir-223 mrna targets and plasma nucleophosmin 1. ) Quantification of RhoB mrna levels (percent control). mir-223 overexpression in human hepatocytes (Huh7) reduces RhoB mrna levels. Mock (n=3), control nonsense mirna (100 nm) (n=3), and mir-223 (100 nm) (n=3). Individual data points, mean +/- s.e.m. b.) Western blot detection of human nucleophosmin 1 (NPM1) protein in Huh7 cell lysates; human plasma; HDL DGUC, HDL isolated by density gradient ultracentrifugation; HDL FPLC, HDL isolated from DGUC > fast protein liquid chromatography; HDL IP, HDL isolated from DGUC > FPLC > antiapoa-i immunoprecipitation; exosomes, and LDL. pab Anti-human NPM1 (1:200); mab Anti-human GAPDH (1:200). ) Human SR-BI (SCARB1) harbors a putative mir-223 target site within its 3 UTR. 4

5 Intracellular mir-375 Fold Change HDL + mir-375 (10 µg ml -1 ) HDL (10 µg ml -1 ) Mifepristone (10 nm) b. SIDT1 mrna (percent control) Intracellular mir-223 (percent control) sisidt1 (100 nm) - + sisidt1 (100 nm) - + Figure S5 HDL-miR-223 delivery is SR-BI-dependent and SIDT1- independent ) Quantification of intracellular hsa-mir-375 levels (fold change). Transfected BHK cells, pswitch human SR-BI inducible (mifepristone 10 nm) expression system, treated with HDL alone (10 µg ml -1 ) (n=3) or HDL+miR-375 (10 µg ml -1 ) (n=3). Data are means +/- s.e.m. b.) Quantification of SIDT1 mrna in human hepatocytes (Huh7). Transient transfection of SIDT1 sirna (sisidt1, 100 nm, SMART-Pool, Dharmacon) (n=11) or control mock transfected cells (n=6). Real-time PCR. RQV, relative quantitative value as normalized by housekeeping gene, peptidylprolyl isomerase A, reported as percent control. Data are means +/- s.e.m. ) Quantification of HDL-miR-223 delivery as determined by intracellular mir-223 levels in human hepatocytes (Huh7). Transient transfection of SIDT1 sirna (sisidt1, 100 nm, SMART-Pool, Dharmacon) (n=3) or control mock transfected cells (n=3). Taqman quantitative PCR, mir-223 standard curve, reported as percent control. Individual data points, means +/- s.e.m. 5

6 Fig.1f b. M r (k) Fig.S4c M r (k) Anti-human CD63 Anti-human NPM1 M r (k) Fig.S4b 17- Anti-human GAPDH Figure S6. Full uncropped Western blot scans. M r (k), molecular weight marker. ) Uncropped Western scan used in Fig.1f. CD63. b.) Uncropped Western scan used in Fig.S4b. NPM1. ) Uncropped Western scan used in Fig.S4b. GAPDH 6