Supplemental Information Appendix: I. Supplemental Methods a. Lipid Analysis b. Proteomics c. Gene Reporter (Luciferase) Assays d. Transcription Factor Binding Assays e. Transcriptomics f. Primers and Probes g. Cell Culture h. HDL-C Uptake Assays i. Cholesterol Biosynthesis Assays j. Cholesterol Efflux Assays k. Animals l. Statistics II. Supplemental Figures a. Figure S1. Cholesterol-depletion reduces miR-223 transcription. b. Figure S2. miR-223 represses HDL-Cholesterol uptake. c. Figure S3. miR-223 represses HDL uptake. d. Figure S4. Predicted miR-223 target sites for human HMGCS1, SC4MOL, SP3, SR- BI, and mouse HMGCS1 and SP3. e. Figure S5. miR-223 over-expression increases HMGCR mRNA levels. SI 1 f. Figure S6. Inhibition of miR-223 increases cholesterol biosynthesis. g. Figure S7. SR-BI expression is increased in low cholesterol states. h. Figure S8. miR-223 repression blocks cholesterol-depletion induced gene expression i. Figure S9. Inhibition of miR-223 does not altered cholesterol efflux. j. Figure S10. miR-223 does not target Sp1 in Huh7 cells. k. Figure S11. Sp3 knockdown results in decreased Sp3 protein in Huh7 cells. l. Figure S12. Sp3 knockdown results in increased ABCA1 mRNA in Huh7 cells. m. Figure S13. Sp3 over-expression results in increased SP3 mRNA in Huh7 cells. n. Figure S14. Sp3 over-expression results in decreased ABCA1 mRNA in Huh7 cells. o. Figure S15. Schematic of Sp1/Sp3 regulation of miR-223’s promoter. p. Figure S16. Plasma lipid levels in miR-223-/- mice. q. Figure S17. SR-BI protein expression in miR-223-/- mouse livers. r. Figure S18. ABCA1 protein expression in miR-223-/- mouse liver. s. Figure S19. Hepatic lipid levels in miR-223-/- mouse liver. t. Figure S20. Inhibition of miR-223 increases triglyceride/ fatty acid biosynthesis. u. Figure S21. Dose response curves for miR-223 regulation. v. Figure S22. miR-223 over-expression in mouse hepatoma cells. w. Figure S23. miR-223 over-expression in human Huh7 hepatoma cells. III. Supplemental Tables a. Table S1. microRNA abundance (rank) in primary hepatocytes, as quantified by real- time PCR-based TaqMan microarrays. SI 2 b. Table S2. Significant differential mRNA expression due to miR-223 over-expression in Huh7 cells. c. Table S3. Predicted (significant) altered transcription factor activity in miR-223 altered gene set. d. Table S4. Significant differential hepatic mRNA expression in miR-223-/- mice. e. Table S5. Significantly up-regulated hepatic mRNAs expression in miR-223-/- mice that are predicted mouse miR-223 target genes. f. Table S6. Significantly up-regulated hepatic mRNAs expression in miR-223-/- mice that are predicted mouse and human miR-223 target genes. g. Table S7. Significantly altered hepatic genes in miR-223-/- that have previously been associated with cholesterol and lipid homeostasis. I. Supplementary Methods a. Lipid Analysis Mouse plasma (300 µL) was injected into an AKTA FPLC (P-920, UPC-900) using a Superose 6 10/300 column (Amersham BioSciences). Total cholesterol and phospholipids were analyzed using Wako Kits, as per manufacturer’s instructions. Triglycerides were quantified using Roche Kits, as per manufacturer’s instructions. Lipids were extracted from mouse livers using the modified Folch extraction method (1). Briefly, 100mg of mouse liver tissue was added to 6 mL of chloroform: methanol (2:1 v/v) followed by the addition of 1.2 mL of 0.05% H2SO4. The sample was then vigorously vortexed and centrifuged. The upper aqueous phase was aspirated off and the volume of the organic phase was recorded. Two sample volumes of 0.01% Trion X- SI 3 100 dissolved in chloroform was added, vigorously vortexed, dried under nitrogen, and then resuspended in 1 mL of diH2O. Total cholesterol and free cholesterol were analyzed using Wako Kits, as per manufacturer’s instructions. Phospholipids were quantified using Wako kits, as per manufacturer’s instructions. Triglycerides were quantified using RaiChem, as per manufacturer’s instructions. b. Proteomics Protein was isolated from washed (2x 1X-Phospahte buffered saline) cells with RIPA buffer (0.05M Hepes, pH 7.6, 1mM EDTA, 0.7% Na-Deoxycholate, 1% NP-40, 0.5M LiCl) containing 1X Complete protease inhibitors (EDTA-free). Lysates were centrifuged for 10 min at 10,000 x g. Total protein (supernatants) were quantified by BCA protein assays (Thermo Scientific). Protein lysates (20 ug) in RIPA buffer were loaded on 4- 12% Bis-Tris gels (Invitrogen) then transferred electrophoretically to nitrocellulose membranes (Invitrogen). After blocking with 5% dry milk in TBST overnight, membranes were incubated for two hours with primary antibodies against Sp3 (1:1000, Santa Cruz), HMGCS1 (1:1000, AbCam), ABCA1 (1:1000, AbCam), SR-B1 (1:5000, Novus), or the loading control PPIA (1:2000, Novus) followed by a two hour incubation with HRP-linked anti-rabbit secondary antibody (1:2000, Santa Cruz) or HRP-linked anti-mouse secondary antibody (1:2000, Santa Cruz). Bands were visualized by incubating with a 50:50 mixture of Western Breeze reagents (PerkinElmer) and exposed to film then developed. Densitometry was carried out using ImageJ software. Western blotting was completed using 20 µg of cleared cell lysate protein. SI 4 c. Gene Reporter and Promoter (Luciferase) Assays The full length 3’ UTRs of SCARB1 (SR-BI), SP3, and HMGCS1 were cloned down- stream of firefly luciferase (Genecopoeia, Inc.). Plasmids (500 ng) were dual-transfected with specific miRNAs and controls (50 nM) for 48 h in HEK293 cells. Cells were lysed and firefly luciferase was normalized to Renilla (transfection control) luciferase. Site- directed mutagenesis was used to generate 3 base deletions for each predicted miR- 223 target site using Stratagene QuickChange XL-II kits and custom primers, as per manufacturer’s instructions. Mutations were confirmed by sequencing. Approximately 3.1kb promoter upstream of miR-223’s transcriptional start site was cloned into a Gluc- ON promoter reporter driving Gaussia luciferase (miR-223 promoter) and secreted alkaline phosphatase (SEAP) as a transfection control (Genecopoeia, Inc.). Promoter reporters were transfected (500ng) in Huh7 cells in FBS 10% DMEM-F12 media for 24 h. Media in specific wells were switched to LPDS %10 DMEM-F12 media for 24 h then switched back to FBS %10 DMEM-F12 media for additional 24 h. 65µL of media was samples at specific time-points to quantify promoter activity in low and normo- cholesterol conditions using Secrete Pair Gaussia Luciferase Assay Kit (Genecopoeia, Inc.). d. Transcription Factor Binding Assays Nuclear extracts were prepared from Huh7 cells and assayed for Sp1 and Sp3 transcription factor binding activity. TransAM Sp1/Sp3 transcriptional factor binding assays were used, as per manufacturer’s instructions (Active Motif). Sp1/Sp3 activity was normalized to total protein concentration of the nuclear fraction. SI 5 e. Transcriptomics Each total RNA pool from the Human Tissue RNA library (Ambion, Inc.) contained total RNA from 3 subjects. Total RNA was isolated from cultured cells or animal tissues using Qiazol and miRNAEasy Kits (Qiagen) or Norgen Total RNA kits, as per protocol. Total RNA was reverse transcribed using TaqMan reverse transcription reagents (Applied Biosystems). Primary miR-223 (pri-mir-223) and all genes of interest (SR-BI, ABCA1, Sp3, HMGCS1, and SC4MOL) were quantified using TaqMan primary miRNA assays or TaqMan mRNA Assays (Applied Biosystems) and normalized to peptidylprolyl isomerase A (PPIA). Real-time PCR for pre-miR-223 was completed using SYBR green and custom primers, and normalized to PPIA. miR-223 and miR-33a were quantified by individual TaqMan miRNA assays (Applied Biosystems) and normalized to U6 (or RNU6). Relative quantitative values were determined using the ΔCt method (RQV = 2- ΔCt). Gene expression analyses in mouse tissues were also completed with the Fluidigm BioMark HD System (96x96). miR-223 (and mock control) was transfected (100nM) into Huh7 cells for 48 h prior to RNA isolation for microarray analysis. Prior to microarray analysis of gene expression, RNA quality was assessed by Agilent 2100 Bioanalyzer (Agilent). Microarray (Affymetrix GeneChip Human Genome U133A 2.0 or Mouse Genome 430_2) studies were completed as previously reported (2). Low-level and high- level analyses were completed using GeneSpring GX12.6 software (Agilent). Raw data were summarized using the LiWong method (3), quantile normalized, and baseline transformed to median of all samples. Raw data were filtered on expression (>20th percentile). Significant (corrected P < 0.05) differential (≥1.5 absolute fold change) gene SI 6 expression changes were determined using unpaired t-tests with multiple testing correction (Benjamini-Hochberg false discovery rate). Normalized gene expression values of miR-223 transfected cells were compared to Mock transfected cell gene expression values, for which have previously been reported (2). MetaCore software (Thomson Reuters) was used for transcription factor enrichment analyses. References to real time PCR analysis, PCR analysis and TaqMan mRNA assays are all the same technique. f. Primers and Probes The following TaqMan primer and probe pairs were used for miRNA analyses; miR-223 002295, miR-29a 2112, U6 001973, RNU6 1093, miR-33a 2135, and control miRNAs (Applied Biosystems). Precursor miR-223 (pre-miR-223) was detected using the following primers; Forward 5’- GCAGTGCCACGCTCCGTGTA-3’, Reverse 5’- TGCCGCACTTGGGGTATTTGACA-3’. The following TaqMan probes were used for gene expression analyses; human PPIA Hu PPIA