Supplemental Information

Appendix:

I. Supplemental Methods a. Lipid Analysis b. Proteomics c. Reporter (Luciferase) Assays d. 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 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 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 . 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 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 . 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. 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 (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 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 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 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). 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 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 FAM-MGB 4333763-0812018, primary miR-223 (pri-mir-223) Hs03303017_pri, human ABCA1 Hs01059118_m1, human Sp3 Hs0595812_mH, human SCARB1 (SR-BI) Hs00969821_m1, human

HMGCS1 Hs00940429_m1, human SC4MOL Hs00198833_m1, human LDLR

Hs00181192_m1, Sp1 Hs00412720_m1, mouse Abca1 Mm00442663_m1, mouse Sp3

Mm00803425_m1, mouse Scarb1 Mm00450234_m1, mouse Hmgcs1

Mm00524111_m1, mouse Sc4mol Mm00499386_m1, mouse Hmgcr Mm01282499_m1, mouse Ldlr Mm00440169_m1, mouse Ppia Mm02342429_g1 (Applied Biosystems).

SI 7 g. Cell culture

Human coronary artery endothelial cells (HCAEC) were cultured in MesoEndo basal cell growth media with growth supplement (Cell Applications, Inc.). Human hepatocytes

(Huh7 cells) and J774 macrophages were cultured in DMEM-F12, 10% fetal bovine serum, streptomycin (100µg/mL), penicillin (100U/mL), and maintained at 37oC with 5%

5 CO2. All cells were plated at 1 x 10 cells/mL for 24h prior to transient transfections for

48 or 72h. miR-223 mimetics (Dharmacon, Inc. miRIDIAN microRNA hsa-miR-223-3p mimic; Accession: MIMAT0000280; C-300580-07-0005 5 nmol.), control miRNA mimetics, control siRNAs, Sp3 siRNA, and SR-BI siRNA were transiently transfected

(50 or 100nM) into human hepatocytes (Huh7) or endothelial cells (HCAEC) using

DharmaFECT4 (All reagents from Dharmacon, Inc.). The mimetic transfection concentrations (100nM) were chosen based on dose response curves for SR-BI,

ABCA1, and SC4MOL, as determined by real-time PCR (Si Appendix Figure S22). miR-

223 is not predicted to target SR-BI mRNA in mice, and over-expression of miR-223

(100nM) in mouse Fl83b hepatoma cells did not result in decreased SR-BI mRNA levels

(SI Appendix Figure S23) HCAEC transfections were carried out under serum-free conditions in MesoEndo growth media (Millipore). Sp3 open reading frame (orf) expression was completed using 0.1 or 100ng of plasmid in Huh7 cells (Genecopoeia,

Inc.). J774 and Huh7 cells were treated with Simvastatin (5µM) or LDL (100 µg/mL total

LDL protein) for 24 h. Over-expression of miR-223 at 100nM resulted in an approximate

40,000–fold increase in cellular miR-223 levels (SI Appendix Fig.S24).

h. HDL-C Uptake Assay

SI 8

HDL-C uptake assays were completed in Huh7 cells. Briefly, Huh7 cells were transfected with miRNAs, HI-223, or controls for 48 or 72h. Cells were washed (2X) in

1X PBS. Cells were treated with DMEM + 0.2% fatty-acid free bovine serum albumin

(BSA) with 3H-cholesteryl ester (Cholesteryl [1,2-3H] hexadecyl ether) HDL (10µg/mL total HDL protein) +/- cold HDL (200µg/mL total HDL protein). HDL was labeled as previously described (4). Cells were incubated with radiolabeled HDL for 4h and media was removed and cells were washed 4X with 1X PBS. Cells were lysed in 0.1% sodium dodecyl sulfate and 0.1N NaOH, scraped, collected, and counted for using a Beckman

LS6000TA/IC Scintillation Counter. HDL uptake counts (D.P.M.) in the presence of excess cold HDL were considered passive uptake and were subtracted from experimental HDL uptake counts (Hot only-(Hot+Cold)) and reported as ng HDL

(protein).

i. Cholesterol Biosynthesis Assays.

Huh7 cells were transfected with control, miR-223, or HI-223 for 48 or 72h prior to incorporation studies and cells were switched to a low glucose-cholesterol-depleted media at 24h. Cells were incubated with 1µCi 3H-acetic acid per well (0.5µCi/mL) in low glucose cholesterol-depleted media for 6-24h and washed (2x) in 1X PBS. Lipids were extracted with hexane:isopropanol (3:2), transferred to a glass tube, and dried under nitrogen stream. Lipids were resuspended in cold cholesterol:cholesteryl oleate, and free and esterified cholesterol was separated by thin-layer chromatography resolved in petroleum ether: diethyl ether: acetic acid (76:20:1). Total cholesterol (3H-cholesterol

SI 9 and 3H-cholesteryl esters were counted (DPM) on Beckman LS6000TA Scintillation

Counter, and normalized to total cellular protein.

j. Cholesterol Efflux Assays

Huh7 cells were transfected with miRNAs, controls, and miRNA hairpin inhibitors

(100nM) for 48 or 72h. Cells were labeled in complete media with 0.5µCi/mL 3H- cholesterol for 24h and washed 2X with 1X PBS. Cholesterol efflux acceptance media contained DMEM F12 media with 0.1% BSA +/-10µg/mL ApoA-I and efflux was carried out for 18h. Cells were lysed in 0.1% sodium dodecyl sulfate and 0.1N NaOH for 10min then scraped and collected. Both the media and cell lysates were counted (DPM) using a Beckman LS6000TA/IC Scintillation Counter. Percent efflux (media/(media+cellular)) was reported as fold change to mock transfection.

k. Animals

All animal studies were conducted under active National Institutes of Health, National

Heart, and Blood Institute Animal Care and Use Committee Protocols. All animals received humane care according to the NIH "Guide for the Care and Use of Laboratory

Animals". Apolipoprotein E-deficient (Apoe-/-) mice (8 weeks) were purchased (The

Jackson Laboratory) and fed a normal chow diet (4% fat, NIH-31 open chow, Zeigler

Brothers) or Cocoa Butter diet (16% fat, 37% calories from fat, 1.25% cholesterol,

0.125% choline chloride, 0.5% sodium cholate, TD90221, Harlan-Teklad) for four weeks. miR-223-/- mice were a gift from Massachusetts Institute of Technology

(Fernando D. Camargo) and Sanford-Burnham Medical Research Institute (Stuart

SI 10

Lipton and Scott R. McKercher)(12), and were fed a normal chow diet. Total RNA was isolated from 20-100mg of mouse liver using Qiazol and miRNAeasy kits or Norgen

Total RNA kits, as per manufacturer’s instructions. Mice were fasted for 4 h prior to blood collection.

l. Statistics

P-values ≤ 0.05 were considered significant and all results expressed as means +/- standard error. For samples sizes n≥4, Mann-Whitney non=parametric tests (two-tailed) were used to compared two groups; for samples n≤3, unpaired (two-tailed) t-tests were used to compare two groups. When comparing > two groups one-way analysis of variance with Newman-Keuls multiple comparison post-test was used.

SI 11

II. Supplemental Figures

a. Figure S1.

Figure S1. Cholesterol-depletion reduces miR-223 transcription. Simvastatin (5µM), cholesterol-depletion (10% LPDS), LDL (100 µg/mL) treatments in J774 for 24 h. FBS, fetal bovine serum. (A) Hmgcr mRNA levels in J774 macrophages. n=4 (B) Primary miR-223 (pri-mir-223) transcript levels in J774 macrophages after 24 h. n=3-4. Data are mean +/-SEM. Mann-Whitney nonparametric tests. *P<0.05

SI 12 b. Figure S2.

Figure S2. miR-223 represses HDL-Cholesterol uptake. (A) Significant differential gene expression in Huh7 cells transiently transfected with miR-223 (100 nM). Benjamini-Hochberg false discovery rate corrected P<0.05, Absolute fold change > 1.5. N=6. Volcano plot illustrating significant down-regulated and up-regulated genes (black dots). (B) Densitometery of SR-BI and ABCA1 Western blots (72h), normalized to GAPDH. miR-223 (100nM) or mock. n=2. Unpaired t-test. (C) SR-BI mRNA levels in human coronary arterial endothelial cells (HCAEC) at 24 and 48 h, as determined by real-time PCR and TaqMan assays. Mock (24h and 48h, n=7), miR-223 (100nM, 24h and 48h, n=7), HI-223 (100nM, 24h n=7, 48h n=5), control siRNA (24h and 48h, n=4), and siRNA SR-BI (24h and 48h, n=4). Paired t-test.

SI 13 c. Figure S3

Figure S3. miR-223 represses HDL uptake. (A) Human coronary artery endothelial cells were transfected with miR-223 (100nM) or control miRNA for 48h prior to radiolabeled HDL (14C- Cholesteryl ester (CE) HDL) uptake assays. Percent uptake (cell counts/cell + media counts) normalized to total cellular protein. n=6. Mann-Whitney nonparametric test. *P=0.002. (B) HDL-C [3H-Cholesteryl ester (CE)] uptake reported as fold change uptake in Huh7 cells. HI-223 (100nM). n=3. Unpaired Student’s t-test. Data are mean +/-SEM *P<0.05, **P<0.0001. Data are mean +/- SEM

SI 14 d. Figure S4.

SI 15

SCARB1 5' 3' 3'UTR 581 U U U C C U C C A G C C U A A A C U G A C A | | : | | | | | | | | hsa-miR-223 A C C C C A U A A A C U G U U U G A C U G U 3' 5'

Figure S4. Predicted miR-223 target sites for human HMGCS1, SC4MOL, SP3, SR- BI, and mouse HMGCS1 and SP3. 3’ UTR, 3’ untranslated region. Red, miR-223 seed region; blue, miR-223; tan, mRNA. (SCARB1) Blue, miR-223 seed region; green, miR- 223; yellow, mRNA. Also found in Vickers et al. (5).

SI 16 e. Figure S5.

Figure S5. miR-223 over-expression increases HMGCR mRNA levels. 3-hydroxy-3- methylglutaryl-CoA reductase (HMGCR) mRNA levels in Huh7 cells. Mock n=7, miR- 223 n=7, and hairpin inhibitor-223 (HI-223) n=6. Mann-Whitney non-parametric test. Data are mean +/-SEM *P<0.05.

SI 17

f. Figure S6.

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Figure S6. Inhibition of miR-223 increases cholesterol biosynthesis. Human Huh7 hepatoma cells were transfected with hairpin inhibitors against miR-223 (HI-223, 100nM). Radiolabeled acetate incorporation assays [3H-acetic acid], followed by thin- layer chromatography. Total cholesterol counts (free cholesterol + cholesteryl ester counts) reported as fold change. n=6. Data are mean +/- SEM

SI 18

g. Figure S7. e

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Figure S7. SR-BI expression is increased in low cholesterol states. SR-BI (SCARB1) mRNA levels in human Huh7 hepatoma cells in cholesterol-starvation (10% lipoprotein-depleted serum). n=6. Data are mean +/- SEM

SI 19 h. Figure S8.

Figure S8. miR-223 repression blocks cholesterol-depletion induced gene expression. Over-expression of miR-223 (100nM) blocks lipoprotein-depleted serum (LPDS) induced (A) scavenger B1 (SR-BI) mRNA levels, (B) HMGCS1 mRNA levels, and (C) SC4MOL mRNA levels in Huh7 cells at 48h, as quantified by real-time PCR TaqMan assays n=2-3. Unpaired t-test. Data are mean +/-SEM *P<0.05.

SI 20 i. Figure S9.

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Figure S9. Inhibition of miR-223 does not altered cholesterol efflux. Human Huh7 hepatoma cells were transfected with hairpin inhibitors against miR-223 (HI-223, 100nM). Cells were labeled with 3H-cholesterol for 24 h prior to efflux to lipid-free apollipoproein A-I (apoA-I). Percent efflux (media counts/cell + media counts) normalized to total cellular protein. n=6. Data are mean +/- SEM

SI 21 j. Figure S10.

Figure S10. miR-223 does not target Sp1 in Huh7 cells. Sp1 mRNA levels in Huh7 cells, as determined by microarray analysis (Mock n=6, miR-223 n=5) and real-time PCR analysis and TaqMan assays (Mock n=4, miR-223 n=4, and hairpin inhibitor (HI- 223) n=4). Data are mean +/-SEM.

SI 22

k. Figure S11

Figure S11. Sp3 knockdown results in decreased Sp3 protein in Huh7 cells. (A) Sp3 protein levels after siRNA knockdown (siRNA Sp3 100nM), as determined by Western blotting. (B) Densitometry of Sp3 protein Western blots. n=3. Unpaired Student’s t-test. *P<0.05 Data are mean +/-SEM.

SI 23 l. Figure S12.

Figure S12. Sp3 knockdown results in increased ABCA1 mRNA in Huh7 cells. ABCA1 mRNA levels after siRNA knockdown of Sp3 in Huh7 cells, as quantified by real-time PCR and TaqMan assays. Mock n=10 and siSRNA Sp3 n=12. Mann-Whitney nonparametric tests. *P<0.05 Data are mean +/-SEM.

SI 24 m. Figure S13

*P = 0.04 5

4

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(mRNA) 2

Fold Change

Sp3 1

0 0 0.1 100 ng SP3

Figure S13. Sp3 over-expression results in increased SP3 mRNA in Huh7 cells. SP3 open reading frame expression construct was transiently transfected (0, 0.1, and 100 ng/mL) into Huh7 cells for 48 h. mRNA levels quantified by real-time PCR (TaqMan). Relative quantitative value (normalized to PPIA) reported as fold change. n=3 Unpaired t-test. Data are mean +/- SEM.

SI 25 n. Figure S14.

Figure S14. Sp3 over-expression results in decreased ABCA1 mRNA in Huh7 cells. ABCA1 mRNA levels in Huh7 cells with Sp3 over-expression, as determined by real-time PCR and TaqMan assays. 0ng n=3, 0.1ng n=3, and 100ng n=3. Unpaired t- test. **P<0.0001 Data are mean +/-SEM.

SI 26 o. Figure S15

Figure S15. Schematic of Sp1/Sp3 regulation of miR-223’s promoter. A, red, predicted Sp3 only transcription factor in miR-223’s promoter. B, blue, predicted Sp1/Sp3 dual transcription factor binding site in miR-223’s promoter. Tss, transcriptional start site.

SI 27 p. Figure S16.

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Figure S16. miR-223-deficiency results in increased Hmgcs1 mRNA expression. Hmgcs1 mRNA levels in miR-223-/- livers compared to WT mice, as determined by microarray (n=6) and real-time PCR and TaqMan assays. WT n=20, miR-223-/- n=27. Mann-Whitney non-parametric test. *P<0.05 Data are mean +/-SEM.

SI 28 q. Figure S17

Figure S17. Plasma lipid levels in miR-223-/- mice. (A) Plasma triglycerides (mg/dL) in miR-223-/- and WT mice. WT n=4, miR-223-/- n=21. Mann-Whitney nonparametric tests. (B) Plasma phospholipids (mg/dL) in miR-223-/- and WT mice. WT n=4, miR-223-/- n=21. Mann-Whitney nonparametric tests. *P<0.05 Data are mean +/-SEM.

SI 29 r. Figure S18

Figure S18. SR-BI protein expression in miR-223-/- mouse livers. A) Western blotting of SR-BI protein in miR-223-/- and WT liver. PPIA loading control. B) Densitometry of SR-BI protein Western blots. n=8. Mann-Whitney non-parametric test. *P<0.05 Data are mean +/-SEM.

SI 30 s. Figure S19

Figure S19. ABCA1 protein expression in miR-223-/- mouse liver. Western blotting of ABCA1 protein in miR-223-/- and WT liver.

SI 31 t. Figure S20.

Figure S20. Hepatic lipid levels in miR-223-/- mouse liver. (A) Hepatic triglyceride levels (mg/dL) in miR-223-/- and WT mice, as quantified by colormetric assays. n=9-10. (B) Hepatic phospholipid levels (mg/dL) in miR-223-/- and WT mice, as quantified by colormetric assays. n=9-10 Data are mean +/-SEM.

SI 32 u. Figure S21.

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Figure S21. Inhibition of miR-223 increases triglyceride/ fatty acid biosynthesis. Human Huh7 hepatoma cells were transfected with hairpin inhibitors against miR-223 (HI-223, 100nM). Radiolabeled acetate incorporation assays [3H-acetic acid], followed by thin-layer chromatography (TLC). Total triglyceride/fatty acid counts (collected TLC plate area between free cholesterol and cholesteryl ester markers, no triglyceride/fatty acid markers) reported as fold change. n=6. Data are mean +/- SEM

SI 33

v. Figure S22

Figure S22. Dose response curves for miR-223 regulation. (A) ABCA1, (B) SC4MOL, and (C) SR-BI mRNA levels in Huh7 cells after 48 h transfection of miR-223 mimetics, as determined by real-time PCR and TaqMan assays. Relative quantitative value (PPIA) reported as fold change to mock transfections. n=4-7 One-Way ANOVA Tukey's multiple comparisons test. Data are mean +/- SEM

SI 34

w. Figure S23.

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Figure S23. miR-223 over-expression in mouse hepatoma cells. SR-BI mRNA levels in mouse Fl83b cells after 48 h transfection of miR-223 mimetics, as determined by real-time PCR and TaqMan assays. Relative quantitative value (PPIA) reported as fold change to mock transfections. n=6. Data are mean +/- SEM

SI 35

x. Figure S24.

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Figure S24. miR-223 over-expression in human Huh7 hepatoma cells. miR-223 levels in human Huh7 cells after 48 h transfection of miR-223 mimetics at 100nM, as determined by real-time PCR and TaqMan assays. Relative quantitative value (U6) reported as fold change to mock transfections. n=4. Data are mean +/- SEM

SI 36

III. Supplemental Tables.

Table S1. microRNA abundance (rank) in primary hepatocytes, as quantified by real-time PCR-based TaqMan microarrays. RQV, relative quantitative value, as normalized to U6.

Rank miRNA RQV 1 hsa-miR-122 0.428317 2 hsa-miR-19b 0.20928 3 hsa-miR-24 0.107129 4 hsa-miR-17 0.053589 5 hsa-miR-192 0.053253 6 hsa-miR-106a 0.053225 7 hsa-miR-146b- 0.053214 5p 8 hsa-miR-30c 0.053125 9 hsa-miR-21 0.052691 10 hsa-miR-30b 0.052434 11 hsa-miR-30a-5p 0.028011 12 hsa-miR-29a 0.027062 13 hsa-miR-125b 0.026898 14 hsa-miR-148a 0.026791 15 hsa-miR-20a 0.026701 16 hsa-miR-26a 0.026676 17 hsa-miR-191 0.026619 18 hsa-miR-194 0.026525 19 hsa-miR-193b 0.026498 20 hsa-miR-526b 0.013931 21 hsa-miR-484 0.013511 22 hsa-miR-99a 0.013288 23 hsa-let-7e 0.013269 24 hsa-miR-100 0.01322 25 hsa-miR-885-5p 0.013187 26 hsa-miR-30a-3p 0.006896 27 hsa-miR-30e-3p 0.006804 28 hsa-miR-26b 0.006764 29 hsa-miR-30d 0.006753 30 hsa-miR-92a 0.006697 31 hsa-let-7g 0.006694 32 hsa-miR-16 0.006685 33 hsa-miR-19a 0.006658 34 hsa-miR-342-3p 0.006654 35 hsa-miR-331-3p 0.006638

SI 37

36 hsa-let-7b 0.006592 37 hsa-miR-22 0.006579 38 hsa-miR-663B 0.003432 39 hsa-miR-378 0.003428 40 hsa-miR-483-5p 0.003407 41 hsa-miR-210 0.003397 42 hsa-let-7c 0.003381 43 hsa-miR-518f 0.003379 44 hsa-miR-28-3p 0.00337 45 hsa-miR-140-5p 0.003352 46 hsa-let-7a 0.003352 47 hsa-miR-320 0.003315 48 hsa-miR-27b 0.003308 49 hsa-miR-126 0.003306 50 hsa-miR-365 0.003276 51 hsa-miR-151-3p 0.001711 52 hsa-miR-192# 0.001701 53 hsa-miR-1233 0.001693 54 hsa-miR-1201 0.001688 55 hsa-miR-151-5P 0.001685 56 hsa-miR-222 0.001684 57 hsa-miR-195 0.001683 58 hsa-miR-106b 0.001681 59 hsa-miR-664 0.00168 60 hsa-miR-455-3p 0.001675 61 hsa-miR-93 0.001672 62 hsa-miR-1275 0.001671 63 hsa-miR-15b 0.00167 64 hsa-miR-455-5p 0.001668 65 hsa-miR-101 0.001667 66 hsa-miR-374b 0.001667 67 hsa-miR-374a 0.001667 68 hsa-miR-34a 0.001666 69 hsa-miR-152 0.001664 70 hsa-miR-20b 0.001661 71 hsa-miR-375 0.00166 72 hsa-miR-186 0.001658 73 hsa-miR-203 0.001657 74 hsa-let-7d 0.00165 75 hsa-miR-103 0.001647 76 hsa-miR-28-5p 0.001645 77 hsa-miR-29c 0.001644 78 hsa-miR-23b 0.001644 79 hsa-miR-22# 0.00085 80 hsa-miR-483-3p 0.000842

SI 38

81 hsa-miR-25 0.000841 82 hsa-miR-130a 0.000838 83 hsa-let-7f 0.000838 84 hsa-miR-339-5p 0.000837 85 hsa-miR-660 0.000833 86 hsa-miR-1260 0.000832 87 hsa-miR-340 0.000831 88 hsa-miR-130b 0.000831 89 hsa-miR-574-3p 0.00083 90 hsa-miR-532-5p 0.000828 91 hsa-miR-328 0.000826 92 hsa-miR-425 0.000823 93 hsa-miR-193a- 0.000823 5p 94 hsa-miR-345 0.000819 95 hsa-miR-885-3p 0.000791 96 hsa-miR-1247 0.000439 97 hsa-miR-1291 0.000434 98 hsa-miR-1290 0.000426 99 hsa-miR-320B 0.000424 100 hsa-miR-223 0.000421 101 hsa-miR-185 0.00042 102 hsa-miR-766 0.00042 103 hsa-miR-145 0.000417 104 hsa-miR-423-5p 0.000416 105 hsa-miR-324-3p 0.000416 106 hsa-miR-29b 0.000416 107 hsa-miR-744 0.000415 108 hsa-miR-204 0.000415 109 hsa-miR-181a 0.000413 110 hsa-miR-146a 0.000413 111 hsa-miR-18b 0.000412 112 hsa-miR-197 0.000411 113 hsa-miR-532-3p 0.000411 114 hsa-miR-15a 0.000407 115 hsa-miR-650 0.00022 116 hsa-miR-769-5p 0.000217 117 hsa-miR-592 0.000214 118 hsa-miR-126# 0.000213 119 hsa-miR-99a# 0.000213 120 hsa-miR-93# 0.000213 121 hsa-miR-20a# 0.000212 122 hsa-miR-149 0.000211 123 hsa-miR-98 0.000211 124 hsa-miR-221 0.00021

SI 39

125 hsa-miR-194# 0.00021 126 hsa-miR-324-5p 0.000209 127 hsa-miR-122# 0.000209 128 hsa-miR-15b# 0.000209 129 hsa-miR-590-3P 0.000209 130 hsa-miR-125b- 0.000209 2# 131 hsa-miR-193b# 0.000209 132 hsa-miR-27b# 0.000209 133 hsa-miR-224 0.000208 134 hsa-miR-106b# 0.000208 135 hsa-miR-361-5p 0.000207 136 hsa-miR-505 0.000207 137 hsa-miR-200b 0.000207 138 hsa-miR-454 0.000207 139 hsa-miR-335 0.000207 140 hsa-miR-661 0.000207 141 hsa-miR-132 0.000206 142 hsa-miR-590-5p 0.000206 143 hsa-miR-18a 0.000206 144 hsa-miR-107 0.000205 145 hsa-miR-27a 0.000205 146 hsa-miR-30d# 0.000203 147 hsa-miR-148b 0.000202 148 hsa-miR-361-3p 0.000113 149 hsa-miR-638 0.000111 150 hsa-miR-1303 0.000108 151 hsa-miR-1285 0.000108 152 hsa-miR-29a# 0.000107 153 hsa-miR-34a# 0.000107 154 hsa-miR-130b# 0.000107 155 hsa-miR-335# 0.000106 156 hsa-miR-1271 0.000106 157 hsa-miR-26b# 0.000106 158 hsa-miR-362-5p 0.000106 159 hsa-miR-19b-1# 0.000105 160 hsa-miR-181a- 0.000105 2# 161 hsa-miR-1248 0.000105 162 hsa-miR-340# 0.000105 163 hsa-miR-142-3p 0.000105 164 hsa-miR-491-5p 0.000104 165 hsa-miR-675 0.000104 166 hsa-miR-200a 0.000103 167 hsa-miR-95 0.000103

SI 40

168 hsa-miR-339-3p 0.000103 169 hsa-miR-296-5p 0.000103 170 hsa-miR-128 0.000103 171 hsa-miR-139-5p 0.000103 172 hsa-miR-214 0.000103 173 hsa-miR-505# 0.000103 174 hsa-miR-200c 9.99E-05 175 hsa-miR-564 5.46E-05 176 hsa-miR-629 5.37E-05 177 hsa-miR-1255B 5.34E-05 178 hsa-miR-621 5.34E-05 179 hsa-miR-571 5.34E-05 180 hsa-miR-148b# 5.33E-05 181 hsa-miR-1180 5.33E-05 182 hsa-miR-17# 5.3E-05 183 hsa-miR-378 5.29E-05 184 hsa-miR-218 5.28E-05 185 hsa-miR-625# 5.28E-05 186 hsa-miR-33a# 5.27E-05 187 hsa-miR-744# 5.26E-05 188 hsa-miR-622 5.24E-05 189 hsa-let-7a# 5.22E-05 190 hsa-miR-9 5.22E-05 191 hsa-miR-21# 5.22E-05 192 hsa-miR-362-3p 5.22E-05 193 hsa-miR-597 5.22E-05 194 hsa-miR-140-3p 5.19E-05 195 hsa-miR-501-5p 5.19E-05 196 hsa-miR-500 5.19E-05 197 hsa-miR-10a 5.19E-05 198 hsa-miR-301a 5.16E-05 199 hsa-miR-199a- 5.16E-05 3p 200 hsa-miR-143 5.15E-05 201 hsa-miR-148a# 5.14E-05 202 hsa-miR-494 5.12E-05 203 hsa-miR-18a# 2.68E-05 204 hsa-miR-1244 2.67E-05 205 hsa-miR-1254 2.66E-05 206 hsa-miR-216b 2.65E-05 207 hsa-let-7f-1# 2.65E-05 208 hsa-miR-26a-1# 2.65E-05 209 hsa-miR-425# 2.64E-05 210 hsa-miR-451 2.64E-05 211 hsa-miR-616 2.63E-05

SI 41

212 hsa-miR-15a# 2.63E-05 213 hsa-let-7g# 2.63E-05 214 hsa-miR-150 2.62E-05 215 hsa-miR-628-3p 2.62E-05 216 hsa-miR-16-1# 2.62E-05 217 hsa-miR-497 2.61E-05 218 hsa-miR-92a-1# 2.61E-05 219 hsa-miR-618 2.61E-05 220 hsa-miR-1300 2.61E-05 221 hsa-miR-452 2.61E-05 222 hsa-miR-429 2.61E-05 223 hsa-miR-222# 2.6E-05 224 hsa-miR-941 2.6E-05 225 hsa-miR-146b- 2.6E-05 3p 226 hsa-miR-499-5p 2.59E-05 227 hsa-miR-487a 2.59E-05 228 hsa-miR-301b 2.59E-05 229 hsa-miR-671-3p 2.57E-05 230 hsa-miR-193a- 2.57E-05 3p 231 hsa-miR-579 2.57E-05 232 hsa-miR-32 2.56E-05 233 hsa-miR-652 2.56E-05 234 hsa-miR-370 2.55E-05 235 hsa-miR-99b 2.55E-05 236 hsa-miR-330-3p 1.36E-05 237 hsa-miR-601 1.35E-05 238 hsa-miR-639 1.34E-05 239 hsa-miR-1282 1.34E-05 240 hsa-miR-34b 1.33E-05 241 hsa-miR-374a# 1.32E-05 242 hsa-miR-155 1.32E-05 243 hsa-miR-27a# 1.32E-05 244 hsa-miR-572 1.32E-05 245 hsa-miR-624 1.31E-05 246 hsa-miR-1183 1.31E-05 247 hsa-miR-422a 1.3E-05 248 hsa-miR-29b-2# 1.3E-05 249 hsa-miR-127-3p 1.3E-05 250 hsa-miR-33a 1.3E-05 251 hsa-miR-376c 1.3E-05 252 hsa-miR-502-3p 1.29E-05 253 hsa-miR-219-5p 1.29E-05 254 hsa-miR-1269 1.28E-05

SI 42

255 hsa-miR-216a 1.28E-05 256 hsa-miR-212 1.28E-05 257 hsa-miR-133a 1.27E-05 258 hsa-miR-502-5p 1.27E-05 259 hsa-miR-191# 6.77E-06 260 hsa-miR-100# 6.75E-06 261 hsa-miR-190b 6.7E-06 262 hsa-miR-550 6.7E-06 263 hsa-miR-802 6.69E-06 264 hsa-let-7f-2# 6.65E-06 265 hsa-miR-7-2# 6.63E-06 266 hsa-miR-9# 6.62E-06 267 hsa-miR-181c 6.6E-06 268 hsa-miR-942 6.58E-06 269 hsa-miR-1276 6.56E-06 270 hsa-miR-589 6.56E-06 271 hsa-let-7i# 6.53E-06 272 hsa-miR-892b 6.52E-06 273 hsa-miR-875-5p 6.51E-06 274 hsa-miR-1262 6.5E-06 275 hsa-miR-649 6.5E-06 276 hsa-miR-1227 6.49E-06 277 hsa-miR-219-1- 6.49E-06 3p 278 hsa-miR-509-5p 6.48E-06 279 hsa-miR-636 6.48E-06 280 hsa-miR-598 6.46E-06 281 hsa-miR-190 6.42E-06 282 hsa-miR-628-5p 6.39E-06 283 hsa-miR-1226# 6.36E-06 284 hsa-miR-576-3p 6.31E-06 285 hsa-miR-545# 3.36E-06 286 hsa-miR-202 3.32E-06 287 hsa-miR-136# 3.31E-06 288 hsa-miR-214# 3.29E-06 289 hsa-miR-548J 3.28E-06 290 hsa-miR-651 3.27E-06 291 hsa-miR-545 3.26E-06 292 hsa-miR-551b 3.25E-06 293 hsa-miR-708 3.25E-06 294 hsa-miR-520c- 3.25E-06 3p 295 hsa-miR-101# 3.25E-06 296 hsa-miR-409-3p 3.24E-06 297 hsa-miR-450b- 3.24E-06

SI 43

5p 298 hsa-miR-411 3.23E-06 299 hsa-miR-548b- 3.23E-06 5p 300 hsa-miR-141 3.23E-06 301 hsa-miR-511 3.23E-06 302 hsa-miR-548d- 3.22E-06 5p 303 hsa-miR-142-5p 3.22E-06 304 hsa-miR-26a-2# 3.22E-06 305 hsa-miR-376a 3.21E-06 306 hsa-miR-616 3.2E-06 307 hsa-miR-656 3.19E-06 308 hsa-miR-33b 3.17E-06 309 hsa-miR-363 3.17E-06 310 hsa-miR-379 3.16E-06 311 hsa-miR-199a- 3.16E-06 5p 312 hsa-miR-604 3.15E-06 313 hsa-miR-519b- 1.76E-06 3p 314 hsa-miR-617 1.67E-06 315 hsa-miR-520D- 1.67E-06 3P 316 hsa-miR-485-3p 1.67E-06 317 hsa-miR-516-3p 1.66E-06 318 hsa-miR-221# 1.66E-06 319 hsa-miR-374b# 1.66E-06 320 hsa-miR-19a# 1.66E-06 321 hsa-miR-125b- 1.65E-06 1# 322 hsa-miR-551b# 1.65E-06 323 hsa-miR-550 1.64E-06 324 hsa-miR-144# 1.63E-06 325 hsa-miR-454# 1.63E-06 326 hsa-miR-137 1.62E-06 327 hsa-miR-582-3p 1.62E-06 328 hsa-miR-548c- 1.62E-06 5p 329 hsa-miR-630 1.61E-06 330 hsa-miR-758 1.61E-06 331 hsa-miR-489 1.61E-06 332 hsa-miR-449a 1.61E-06 333 hsa-miR-576-5p 1.61E-06 334 hsa-miR-302a 1.6E-06 335 hsa-miR-135b 1.6E-06

SI 44

336 hsa-miR-539 1.6E-06 337 hsa-miR-523 1.59E-06 338 hsa-miR-323-3p 1.59E-06 339 hsa-miR-495 1.57E-06 340 hsa-miR-223# 8.3E-07 341 hsa-miR-184 8.27E-07 342 hsa-miR-645 8.26E-07 343 hsa-miR-548d- 8.25E-07 3p 344 hsa-miR-125a- 8.17E-07 3p 345 hsa-miR-200a# 8.17E-07 346 hsa-miR-130a# 8.15E-07 347 hsa-miR-570 8.14E-07 348 hsa-miR-542-3p 8.13E-07 349 hsa-miR-625 8.12E-07 350 hsa-miR-641 8.12E-07 351 hsa-miR-586 8.08E-07 352 hsa-miR-655 8.07E-07 353 hsa-miR-125a- 8.05E-07 5p 354 hsa-miR-493 8.05E-07 355 hsa-miR-338-3p 8.02E-07 356 hsa-miR-580 7.95E-07 357 hsa-miR-141# 7.92E-07 358 hsa-miR-629 7.86E-07 359 hsa-miR-183 7.72E-07 360 hsa-miR-635 4.14E-07 361 hsa-miR-888 4.11E-07 362 hsa-miR-145# 4.06E-07 363 hsa-miR-488 4.06E-07 364 hsa-miR-519e 4.03E-07 365 hsa-miR-372 4.02E-07 366 hsa-miR-410 3.99E-07 367 hsa-miR-331-5p 3.97E-07 368 hsa-miR-338-5P 2.15E-07 369 hsa-miR-548P 2.11E-07 370 hsa-miR-548E 2.08E-07 371 hsa-miR-548I 2.08E-07 372 hsa-miR-10a# 2.08E-07 373 hsa-miR-518a- 2.07E-07 3p 374 hsa-miR-548K 2.07E-07 375 hsa-miR-1256 2.06E-07 376 hsa-miR-1302 2.05E-07 377 hsa-miR-337-5p 2.05E-07

SI 45

378 hsa-miR-138 2.04E-07 379 hsa-miR-944 2.04E-07 380 hsa-miR-380-5p 2.03E-07 381 hsa-miR-135a 2.03E-07 382 hsa-miR-517c 2.03E-07 383 hsa-miR-627 2.03E-07 384 hsa-miR-424 2.02E-07 385 hsa-miR-1179 2.02E-07 386 hsa-miR-182 2E-07 387 hsa-miR-302a# 1.99E-07 388 hsa-miR-517b 1.99E-07 389 hsa-miR-34c-5p 1.99E-07 390 hsa-miR-519a 1.99E-07 391 hsa-miR-922 1.92E-07 392 hsa-miR-561 1.92E-07 393 hsa-miR-1243 9.81E-08 394 hsa-miR-1 9.76E-08

Table S2. Significant differential mRNA expression due to miR-223 over- expression in Huh7 cells (Benjamini-Hochberg Corrected P-value < 0.05; >1.5 absolute fold change).

Gene Fold P-Value Corrected Gene Title RefSeq Symbol Change P-Value Gene Transcript ID PAPOLA -7.85 9.58E-04 1.55E-02 poly(A) alpha 10914 NM_032632

CD44 -7.58 1.38E-03 1.93E-02 CD44 molecule (Indian blood group) 960 NM_000610

TRIM25 -4.83 1.64E-03 2.14E-02 tripartite motif-containing 25 7706 NM_005082

KHSRP -4.65 6.20E-03 4.74E-02 KH-type splicing regulatory protein 8570 NM_003685

HLF -3.35 9.17E-04 1.54E-02 hepatic leukemia factor 3131 NM_002126

ARRB1 -3.23 2.27E-04 7.71E-03 , beta 1 408 NM_004041

TRIM14 -2.71 2.91E-05 3.22E-03 tripartite motif-containing 14 9830 NM_014788

NUP210 -2.59 2.77E-06 1.40E-03 210kDa 23225 NM_024923

CDX2 -2.50 2.40E-04 7.98E-03 caudal type 2 1045 NM_001265

RCN2 -2.42 7.27E-05 4.75E-03 reticulocalbin 2, EF-hand calcium binding domain 5955 NM_002902

NF2 -2.42 5.76E-03 4.53E-02 neurofibromin 2 () 4771 NM_000268

LPAR2 -2.38 2.93E-03 3.01E-02 lysophosphatidic acid receptor 2 9170 NM_004720

GLYR1 -2.37 1.02E-06 1.30E-03 glyoxylate reductase 1 homolog (Arabidopsis) 23157 NM_015129

DPM2 -2.34 4.33E-06 1.60E-03 dolichyl-phosphate mannosyltransferase polypeptide 2, 8818 NM_003863 regulatory subunit PBK -2.34 4.39E-04 1.06E-02 PDZ binding kinase 55872 NM_018492

GLYR1 -2.32 9.81E-05 5.38E-03 glyoxylate reductase 1 homolog (Arabidopsis) 84656 NM_032569

SCARB1 -2.31 4.87E-03 4.06E-02 scavenger receptor class B, member 1 949 NM_005505

TNFRSF12A -2.30 3.74E-06 1.60E-03 tumor necrosis factor receptor superfamily, member 12A 51330 NM_016639

MAP2K6 -2.28 4.81E-03 4.04E-02 mitogen-activated protein kinase kinase 6 5608 NM_002758

SI 46

PARP1 -2.24 1.67E-05 2.51E-03 poly (ADP-ribose) polymerase 1 142 NM_001618

CYB5A -2.23 3.00E-03 3.06E-02 cytochrome b5 type A (microsomal) 1528 NM_001914

SEPT2 -2.18 1.25E-05 2.31E-03 2 4735 NM_004404

CBX5 -2.17 3.27E-06 1.45E-03 chromobox homolog 5 23468 NM_012117

MDM2 -2.16 7.80E-04 1.42E-02 binding protein homolog (mouse) 4193 NM_002392

LOX -2.15 2.15E-04 7.49E-03 lysyl oxidase 4015 NM_002317

ARHGDIA -2.14 1.20E-05 2.31E-03 Rho GDP dissociation inhibitor (GDI) alpha 396 NM_004309

C19orf6 -2.13 2.81E-04 8.57E-03 19 open reading frame 6 91304 NM_033420

C4BPB -2.13 1.60E-06 1.37E-03 complement component 4 binding protein, beta 725 NM_000716

MDK -2.11 1.04E-05 2.24E-03 midkine (neurite growth-promoting factor 2) 4192 NM_002391

SLC23A2 -2.11 2.43E-06 1.40E-03 solute carrier family 23 (nucleobase transporters), member 2 9962 NM_005116

SLC19A1 -2.06 7.08E-08 6.64E-04 solute carrier family 19 (folate transporter), member 1 6573 NM_194255

TBC1D17 -2.06 1.05E-04 5.61E-03 TBC1 domain family, member 17 79735 NM_024682

WWTR1 -2.05 2.88E-07 1.13E-03 WW domain containing transcription regulator 1 25937 NM_015472

SLMO2 -2.02 3.08E-05 3.36E-03 slowmo homolog 2 () 51012 NM_016045

TMEM158 -2.01 3.81E-05 3.75E-03 transmembrane protein 158 (gene/pseudogene) 25907 NM_015444

AK4 -1.99 1.55E-05 2.39E-03 adenylate kinase 4 205 NM_013410

SEPT10 -1.98 4.06E-05 3.75E-03 septin 10 151011 NM_144710

CCND2 -1.97 4.39E-03 3.82E-02 cyclin D2 894 NM_001759

E2F1 -1.97 1.46E-06 1.37E-03 transcription factor 1 1869 NM_005225

COPS8 -1.96 3.97E-05 3.75E-03 COP9 constitutive photomorphogenic homolog subunit 8 10920 NM_006710 (Arabidopsis) RBFOX2 -1.96 2.78E-04 8.57E-03 RNA binding protein, fox-1 homolog (C. elegans) 2 23543 NM_014309

STMN1 -1.95 2.30E-04 7.77E-03 1 3925 NM_005563

GREB1 -1.95 1.84E-05 2.62E-03 growth regulation by estrogen in breast cancer 1 9687 NM_014668

SEPT6 -1.94 2.24E-06 1.40E-03 septin 6 23157 NM_145799

TRIM31 -1.92 1.04E-04 5.58E-03 tripartite motif-containing 31 11074 NM_007028

NQO1 -1.91 3.58E-05 3.66E-03 NAD(P)H dehydrogenase, quinone 1 1728 NM_000903

SLC11A2 -1.90 1.04E-04 5.57E-03 solute carrier family 11 (proton-coupled divalent metal ion 4891 NM_000617 transporters), member 2 C18orf10 -1.89 2.21E-03 2.55E-02 open reading frame 10 25941 NM_015476

SLC16A3 -1.88 4.16E-04 1.04E-02 solute carrier family 16, member 3 (monocarboxylic acid 9123 NM_004207 transporter 4) CDKN2C -1.88 5.50E-03 4.39E-02 cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4) 1031 NM_001262

GAMT -1.87 1.35E-03 1.92E-02 guanidinoacetate N-methyltransferase 2593 NM_000156

SSTR2 -1.86 1.10E-03 1.67E-02 somatostatin receptor 2 6752 NM_001050

SLC39A8 -1.85 1.10E-05 2.28E-03 solute carrier family 39 (zinc transporter), member 8 64116 NM_022154

SLC29A1 -1.85 4.10E-03 3.67E-02 solute carrier family 29 (nucleoside transporters), member 1 2030 NM_004955

IL24 -1.85 1.35E-03 1.92E-02 interleukin 24 11009 NM_006850

WBSCR22 -1.84 4.18E-06 1.60E-03 Williams Beuren syndrome chromosome region 22 114049 NM_017528

AKR7A2 -1.84 1.17E-04 5.81E-03 aldo-keto reductase family 7, member A2 (aflatoxin aldehyde 8574 NM_003689 reductase) SMARCD1 -1.83 7.43E-07 1.30E-03 SWI/SNF related, matrix associated, dependent 6602 NM_003076 regulator of , subfamily d, member 1 ATP1B1 -1.83 1.42E-06 1.37E-03 ATPase, Na+/K+ transporting, beta 1 polypeptide 481 NM_001677

PKNOX1 -1.82 1.32E-04 6.09E-03 PBX/knotted 1 homeobox 1 5316 NM_004571

MAD1L1 -1.81 5.86E-04 1.21E-02 MAD1 mitotic arrest deficient-like 1 (yeast) 8379 NM_003550

BIRC5 -1.80 2.43E-04 7.99E-03 baculoviral IAP repeat-containing 5 332 NM_001168

SI 47

DBN1 -1.80 1.58E-07 7.74E-04 drebrin 1 1627 NM_004395

DCXR -1.79 3.68E-04 9.82E-03 dicarbonyl/L-xylulose reductase 51181 NM_016286

CENPN -1.78 5.98E-05 4.52E-03 protein N 55839 NM_018455

PRKACA -1.78 1.87E-05 2.63E-03 protein kinase, cAMP-dependent, catalytic, alpha 5566 NM_002730

TFPT -1.78 8.06E-04 1.44E-02 TCF3 (E2A) fusion partner (in childhood Leukemia) 29844 NM_013342

SLC4A7 -1.77 1.40E-04 6.24E-03 solute carrier family 4, sodium bicarbonate cotransporter, 9497 NM_003615 member 7 PEX19 -1.76 9.85E-05 5.38E-03 peroxisomal biogenesis factor 19 5824 NM_002857

YIPF2 -1.76 5.83E-03 4.57E-02 Yip1 domain family, member 2 78992 NM_024029

GPX3 -1.76 1.91E-04 7.13E-03 glutathione peroxidase 3 (plasma) 2878 NM_002084

HIPK2 -1.76 1.53E-05 2.39E-03 homeodomain interacting protein kinase 2 28996 NM_022740

KLF5 -1.75 3.75E-05 3.75E-03 Kruppel-like factor 5 (intestinal) 688 NM_001730

CISD1 -1.75 6.72E-05 4.63E-03 CDGSH iron sulfur domain 1 55847 NM_018464

CRIP1 -1.74 1.60E-03 2.12E-02 cysteine-rich protein 1 (intestinal) 1396 NM_001311

ALDH4A1 -1.74 1.76E-04 6.95E-03 aldehyde dehydrogenase 4 family, member A1 8659 NM_003748

SNX24 -1.74 1.05E-05 2.24E-03 sorting nexin 24 28966 NM_014035

SLC4A4 -1.73 9.47E-05 5.32E-03 solute carrier family 4, sodium bicarbonate cotransporter, 8671 NM_003759 member 4 BAG2 -1.73 6.28E-06 1.83E-03 BCL2-associated athanogene 2 9532 NM_004282

BMP4 -1.72 7.55E-04 1.40E-02 bone morphogenetic protein 4 652 NM_001202

MRPS17 -1.72 3.97E-05 3.75E-03 mitochondrial ribosomal protein S17 /// protein 713 51373 NM_015969

BOP1 -1.72 1.26E-05 2.31E-03 block of proliferation 1 23246 NM_015201

SARDH -1.72 1.03E-03 1.61E-02 sarcosine dehydrogenase 1757 NM_007101

TWF1 -1.71 4.75E-05 4.01E-03 twinfilin, actin-binding protein, homolog 1 (Drosophila) 5756 NM_002822

TMC6 -1.71 2.63E-03 2.81E-02 transmembrane channel-like 6 11322 NM_007267

DDIT4 -1.70 5.78E-05 4.48E-03 DNA-damage-inducible transcript 4 54541 NM_019058

AKAP1 -1.69 5.56E-04 1.19E-02 A kinase (PRKA) anchor protein 1 8165 NM_003488

ETFB -1.69 6.05E-06 1.80E-03 electron-transfer-flavoprotein, beta polypeptide 2109 NM_001985

MAU2 -1.69 3.74E-03 3.47E-02 MAU2 chromatid cohesion factor homolog (C. elegans) 23383 NM_015329

RECQL -1.69 6.12E-05 4.54E-03 RecQ protein-like (DNA Q1-like) 5965 NM_002907

AGFG2 -1.69 4.87E-04 1.11E-02 ArfGAP with FG repeats 2 3268 NM_006076

SLC39A1 -1.68 9.48E-07 1.30E-03 solute carrier family 39 (zinc transporter), member 1 27173 NM_014437

PEX16 -1.68 2.87E-04 8.58E-03 peroxisomal biogenesis factor 16 9409 NM_004813

POLD2 -1.68 1.49E-06 1.37E-03 polymerase (DNA directed), delta 2, regulatory subunit 5425 NM_006230 50kDa DAG1 -1.68 6.75E-06 1.86E-03 1 (-associated glycoprotein 1) 1605 NM_004393

PGF -1.67 3.16E-06 1.45E-03 5228 NM_002632

RNF126 -1.67 1.40E-04 6.24E-03 ring finger protein 126 55658 NM_194460

SCAMP3 -1.66 5.25E-06 1.71E-03 secretory carrier 3 10067 NM_005698

CNP -1.66 1.83E-05 2.62E-03 2',3'-cyclic nucleotide 3' 1267 NM_033133

UBXN1 -1.66 3.40E-03 3.29E-02 UBX domain protein 1 51035 NM_015853

EIF5A -1.66 5.94E-04 1.21E-02 eukaryotic translation initiation factor 5A 1984 NM_001970

TYRO3 -1.66 4.19E-03 3.72E-02 TYRO3 protein tyrosine kinase 7301 NM_006293

XYLB -1.65 1.62E-03 2.13E-02 xylulokinase homolog (H. influenzae) 9942 NM_005108

DCTN1 -1.65 5.63E-04 1.19E-02 1 1639 NM_004082

PSIP1 -1.64 1.89E-03 2.33E-02 PC4 and SFRS1 interacting protein 1 11168 NM_021144

SI 48

STK39 -1.64 2.11E-05 2.78E-03 serine threonine kinase 39 27347 NM_013233

ARL6IP1 -1.64 1.45E-03 1.99E-02 ADP-ribosylation factor-like 6 interacting protein 1 23204 NM_015161

TSPAN7 -1.64 1.71E-03 2.20E-02 7 7102 NM_004615

AAMP -1.64 1.96E-04 7.22E-03 angio-associated, migratory cell protein 14 NM_001087

GDF11 -1.64 8.19E-04 1.45E-02 growth differentiation factor 11 10220 NM_005811

PHLPP1 -1.63 6.86E-06 1.86E-03 PH domain and leucine rich repeat 1 23239 NM_194449

NDUFA5 -1.63 5.91E-04 1.21E-02 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 5, 4698 NM_005000 13kDa ATP6V0E2 -1.62 3.13E-03 3.13E-02 ATPase, H+ transporting V0 subunit e2 155066 NM_145230

BAZ1B -1.62 8.00E-04 1.43E-02 bromodomain adjacent to zinc finger domain, 1B 9031 NM_032408

FAM115A -1.62 4.14E-07 1.30E-03 family with sequence similarity 115, member A 9747 NM_014719

LYRM4 -1.62 2.44E-05 2.87E-03 LYR motif containing 4 57128 NM_020408

SIGMAR1 -1.62 1.79E-04 7.01E-03 sigma non-opioid 1 10280 NM_005866

CTSL2 -1.62 1.18E-05 2.31E-03 cathepsin L2 1515 NM_001333

AP3D1 -1.61 2.16E-04 7.49E-03 adaptor-related protein complex 3, delta 1 subunit 8943 NM_003938

PITPNA -1.61 2.42E-04 7.99E-03 phosphatidylinositol transfer protein, alpha 5306 NM_006224

PPP4R2 -1.60 4.18E-04 1.04E-02 protein phosphatase 4, regulatory subunit 2 151987 NM_174907

PRPF31 -1.59 1.55E-03 2.07E-02 PRP31 pre-mRNA processing factor 31 homolog (S. 26121 NM_015629 cerevisiae) FEZ2 -1.59 7.43E-05 4.75E-03 fasciculation and elongation protein zeta 2 (zygin II) 9637 NM_005102

LEF1 -1.59 8.67E-05 5.12E-03 lymphoid -binding factor 1 51176 NM_016269

RRP7A -1.59 2.35E-03 2.63E-02 ribosomal RNA processing 7 homolog A (S. cerevisiae) 27341 NM_015703

FYN -1.59 5.33E-04 1.16E-02 FYN oncogene related to SRC, FGR, YES 2534 NM_002037

PKP2 -1.58 1.54E-03 2.07E-02 2 5318 NM_004572

PEX3 -1.58 2.23E-03 2.56E-02 peroxisomal biogenesis factor 3 8504 NM_003630

AHNAK -1.58 1.23E-04 5.91E-03 AHNAK nucleoprotein 79026 NM_001620

SLC7A8 -1.58 1.26E-03 1.83E-02 solute carrier family 7 ( transporter, L-type), 23428 NM_012244 member 8 FOXO1 -1.58 7.97E-07 1.30E-03 forkhead box O1 2308 NM_002015

FKBP1A -1.58 3.62E-03 3.41E-02 FK506 binding protein 1A, 12kDa 2280 NM_000801

TUBB2A -1.57 4.49E-04 1.07E-02 , beta 2A /// tubulin, beta 2B 7280 NM_001069

PPP2R1A -1.57 3.73E-04 9.87E-03 , regulatory subunit A, alpha 5518 NM_014225

EMD -1.57 3.39E-04 9.33E-03 emerin 2010 NM_000117

EXOSC5 -1.57 9.81E-04 1.57E-02 exosome component 5 56915 NM_020158

WDR1 -1.57 5.48E-03 4.38E-02 WD repeat domain 1 9948 NM_005112

TWF2 -1.57 1.19E-04 5.83E-03 twinfilin, actin-binding protein, homolog 2 (Drosophila) 11344 NM_007284

FARSA -1.56 1.66E-04 6.81E-03 phenylalanyl-tRNA synthetase, alpha subunit 2193 NM_004461

KIFC1 -1.56 9.21E-04 1.54E-02 family member C1 3833 NM_002263

HK2 -1.56 7.85E-05 4.86E-03 hexokinase 2 3099 NM_000189

UBE2S -1.56 2.01E-04 7.29E-03 ubiquitin-conjugating E2S 27338 NM_014501

CDT1 -1.56 2.84E-04 8.58E-03 chromatin licensing and DNA replication factor 1 81620 NM_030928

IFT57 -1.56 6.26E-05 4.56E-03 intraflagellar transport 57 homolog (Chlamydomonas) 55081 NM_018010

ZNF22 -1.56 3.94E-03 3.58E-02 zinc finger protein 22 (KOX 15) 7570 NM_006963

ARMCX1 -1.56 2.51E-03 2.74E-02 armadillo repeat containing, X-linked 1 51309 NM_016608

AK2 -1.56 4.01E-06 1.60E-03 adenylate kinase 2 204 NM_001625

COX11 -1.56 5.24E-04 1.15E-02 COX11 cytochrome c oxidase assembly homolog (yeast) 1353 NM_004375

SI 49

SLC43A3 -1.56 3.99E-05 3.75E-03 solute carrier family 43, member 3 29015 NM_014096

NDRG1 -1.56 2.43E-05 2.87E-03 N- downstream regulated 1 10397 NM_006096

C13orf15 -1.55 2.16E-04 7.49E-03 chromosome 13 open reading frame 15 28984 NM_014059

TCF7L2 -1.55 3.98E-04 1.02E-02 transcription factor 7-like 2 (T-cell specific, HMG-box) 6934 NM_030756

TMEM14B -1.55 3.91E-05 3.75E-03 transmembrane protein 14B 81853 NM_030969

NAT8 -1.55 6.40E-03 4.85E-02 N-acetyltransferase 8 (GCN5-related, putative) 9027 NM_003960

CLSTN1 -1.55 8.07E-05 4.96E-03 calsyntenin 1 22883 NM_014944

ETV5 -1.55 1.41E-05 2.39E-03 2119 NM_004454

POLR3G -1.55 7.22E-05 4.75E-03 polymerase (RNA) III (DNA directed) polypeptide G (32kD) 10622 NM_006467

FST -1.55 1.84E-04 7.08E-03 follistatin 10468 NM_006350

IFRD2 -1.55 2.16E-06 1.40E-03 interferon-related developmental regulator 2 7866 NM_006764

DBT -1.54 2.28E-03 2.59E-02 dihydrolipoamide branched chain transacylase E2 1629 NM_001918

ZNF282 -1.54 2.12E-03 2.48E-02 zinc finger protein 282 8427 NM_003575

ACTC1 -1.54 8.34E-04 1.46E-02 actin, alpha, cardiac muscle 1 70 NM_005159

ABCF2 -1.54 4.50E-03 3.88E-02 ATP-binding cassette, sub-family F (GCN20), member 2 10061 NM_005692

DCTD -1.54 6.30E-03 4.79E-02 dCMP deaminase 1635 NM_001921

PPPDE1 -1.54 8.18E-05 4.98E-03 PPPDE peptidase domain containing 1 51029 NM_016076

VDR -1.53 3.01E-04 8.83E-03 vitamin D (1,25- dihydroxyvitamin D3) receptor 7421 NM_000376

ARG2 -1.53 6.53E-04 1.29E-02 arginase, type II 384 NM_001172

RAD23B -1.53 3.60E-03 3.40E-02 RAD23 homolog B (S. cerevisiae) 5887 NM_002874

RWDD1 -1.53 2.01E-05 2.69E-03 RWD domain containing 1 51389 NM_015952

PHTF2 -1.52 6.26E-05 4.56E-03 putative homeodomain transcription factor 2 57157 NM_020432

PCYT2 -1.52 1.92E-05 2.68E-03 phosphate cytidylyltransferase 2, ethanolamine 5833 NM_002861

DAK -1.52 2.91E-03 3.00E-02 dihydroxyacetone kinase 2 homolog (S. cerevisiae) 26007 NM_015533

CCND3 -1.52 8.80E-05 5.14E-03 cyclin D3 896 NM_001760

HAT1 -1.52 4.06E-04 1.03E-02 acetyltransferase 1 8520 NM_003642

DENND5B -1.52 7.19E-05 4.75E-03 DENN/MADD domain containing 5B 160518 NM_144973

FOXD1 -1.52 6.39E-03 4.85E-02 2297 NM_004472

C1orf144 -1.52 5.72E-03 4.51E-02 open reading frame 144 26099 NM_015609

SSRP1 -1.51 4.46E-06 1.61E-03 structure specific recognition protein 1 6749 NM_003146

FEM1B -1.51 1.41E-03 1.95E-02 fem-1 homolog b (C. elegans) 10116 NM_015322

AIM1 -1.51 7.77E-04 1.41E-02 absent in melanoma 1 202 NM_001624

GALR2 -1.51 3.71E-04 9.84E-03 galanin receptor 2 8811 NM_003857

RASSF4 -1.51 1.03E-03 1.61E-02 Ras association (RalGDS/AF-6) domain family member 4 83937 NM_032023

LMO4 -1.51 3.75E-04 9.88E-03 LIM domain only 4 8543 NM_006769

CRLF1 -1.51 7.42E-05 4.75E-03 cytokine receptor-like factor 1 9244 NM_004750

OR7A10 -1.51 2.63E-03 2.82E-02 olfactory receptor, family 7, subfamily A, member 10 390892 NM_001005190

TLE3 -1.51 1.79E-03 2.25E-02 -like enhancer of split 3 (E(sp1) homolog, 7090 NM_005078 Drosophila) CDC27 -1.50 1.63E-04 6.75E-03 cell division cycle 27 homolog (S. cerevisiae) 996 NM_001256

STK38L 1.50 1.22E-04 5.91E-03 serine/threonine kinase 38 like 23012 NM_015000

F3 1.50 5.48E-03 4.38E-02 coagulation factor III (thromboplastin, tissue factor) 2152 NM_001993

FPGT 1.50 1.53E-04 6.54E-03 fucose-1-phosphate guanylyltransferase 8790 NM_003838

ZNF267 1.51 1.14E-03 1.71E-02 zinc finger protein 267 10308 NM_003414

SI 50

RAB23 1.51 2.13E-03 2.49E-02 RAB23, member RAS oncogene family 51715 NM_016277

SPATS2 1.51 1.63E-03 2.13E-02 associated, serine-rich 2 65244 NM_023071

UBA6 1.51 1.19E-03 1.76E-02 ubiquitin-like modifier activating enzyme 6 55236 NM_018227

ASPH 1.51 8.19E-04 1.45E-02 aspartate beta-hydroxylase 444 NM_004318

NAGPA 1.51 1.13E-04 5.76E-03 N-acetylglucosamine-1-phosphodiester alpha-N- 51172 NM_016256 acetylglucosaminidase CXCL2 1.51 2.07E-03 2.45E-02 chemokine (C-X-C motif) ligand 2 2920 NM_002089

VPS41 1.51 2.65E-05 3.09E-03 vacuolar protein sorting 41 homolog (S. cerevisiae) 27072 NM_014396

FAM47E 1.51 8.87E-04 1.51E-02 family with sequence similarity 47, member E 8987 NM_003943

SMEK1 1.52 1.05E-03 1.62E-02 SMEK homolog 1, suppressor of mek1 (Dictyostelium) 55671 NM_032560

SMG7 1.52 9.31E-04 1.54E-02 Smg-7 homolog, nonsense mediated mRNA decay factor (C. 9887 NM_201568 elegans) HMOX1 1.52 4.07E-03 3.65E-02 heme oxygenase (decycling) 1 3162 NM_002133

ADRA2C 1.52 6.08E-05 4.53E-03 adrenergic, alpha-2C-, receptor 152 NM_000683

PCDH9 1.52 2.58E-03 2.79E-02 protocadherin 9 5101 NM_020403

SAMD4A 1.53 4.03E-03 3.63E-02 sterile alpha motif domain containing 4A 23034 NM_015589

ZNF43 1.53 7.95E-06 1.99E-03 zinc finger protein 43 7594 NM_003423

PDGFRA 1.53 1.61E-03 2.12E-02 platelet-derived growth factor receptor, alpha polypeptide 5156 NM_006206

EGOT 1.53 1.87E-03 2.32E-02 eosinophil granule ontogeny transcript (non-protein coding) 100126791 NR_004428

PROZ 1.53 5.84E-04 1.21E-02 protein Z, vitamin K-dependent plasma glycoprotein 8858 NM_003891

LGR5 1.53 4.82E-03 4.04E-02 leucine-rich repeat-containing -coupled receptor 5 8549 NM_003667

CALD1 1.53 6.13E-04 1.23E-02 caldesmon 1 800 NM_004342

ZNF117 1.53 7.40E-05 4.75E-03 zinc finger protein 117 51351 NM_015852

RCHY1 1.53 6.14E-03 4.71E-02 ring finger and CHY zinc finger domain containing 1 25898 NM_015436

CTSH 1.53 1.12E-03 1.69E-02 cathepsin H 1512 NM_004390

FAM21A 1.53 2.86E-05 3.19E-03 family with sequence similarity 21, member A 55747 NM_015262

CX3CL1 1.54 1.15E-04 5.76E-03 chemokine (C-X3-C motif) ligand 1 6376 NM_002996

ACTR2 1.54 1.08E-04 5.68E-03 ARP2 actin-related protein 2 homolog (yeast) 10097 NM_005722

ABCA5 1.54 4.18E-04 1.04E-02 ATP-binding cassette, sub-family A (ABC1), member 5 23461 NM_018672

DHRS7 1.54 1.18E-03 1.75E-02 Dehydrogenase/reductase (SDR family) member 7 51635 NM_016029

TUBA1A 1.54 5.81E-03 4.55E-02 tubulin, alpha 1a 7846 NM_006009

ANKRD12 1.54 1.13E-03 1.71E-02 repeat domain 12 23253 NM_015208

DCUN1D4 1.54 2.94E-04 8.73E-03 DCN1, defective in cullin neddylation 1, domain containing 4 23142 NM_015115 (S. cerevisiae) WDR44 1.54 8.91E-04 1.51E-02 WD repeat domain 44 54521 NM_019045

DTNA 1.55 3.24E-03 3.20E-02 dystrobrevin, alpha 1837 NM_001390

CREBL2 1.55 5.72E-06 1.78E-03 cAMP responsive element binding protein-like 2 1389 NM_001310

CDH2 1.55 4.51E-03 3.88E-02 cadherin 2, type 1, N-cadherin (neuronal) 1000 NM_001792

CYLD 1.55 6.64E-03 4.97E-02 cylindromatosis (turban tumor syndrome) 1540 NM_015247

FILIP1L 1.55 4.90E-04 1.11E-02 A interacting protein 1-like 11259 NM_014890

JUNB 1.55 4.60E-03 3.93E-02 jun B proto-oncogene 3726 NM_002229

LOH3CR2A 1.56 1.95E-03 2.37E-02 loss of heterozygosity, 3, chromosomal region 2, gene A 29931 NR_024065

CLDN1 1.56 4.28E-04 1.05E-02 claudin 1 9076 NM_021101

ATP6V0E1 1.56 3.14E-03 3.14E-02 ATPase, H+ transporting, lysosomal 9kDa, V0 subunit e1 8992 NM_003945

TRIM23 1.56 2.41E-03 2.67E-02 tripartite motif-containing 23 373 NM_001656

STAM2 1.56 1.24E-04 5.91E-03 signal transducing adaptor molecule (SH3 domain and ITAM 10254 NM_005843 motif) 2

SI 51

APOL2 1.57 9.87E-04 1.58E-02 apolipoprotein L, 2 23780 NM_030882

SSBP3 1.57 5.26E-03 4.26E-02 single stranded DNA binding protein 3 23648 NM_018070

FYCO1 1.57 8.99E-04 1.52E-02 FYVE and coiled-coil domain containing 1 79443 NM_024513

TSPAN12 1.58 4.42E-04 1.06E-02 tetraspanin 12 23554 NM_012338

FAM21C 1.58 2.42E-05 2.87E-03 family with sequence similarity 21, member C 253725 NM_015262

CAV2 1.58 2.97E-04 8.77E-03 2 858 NM_001233

SMG1 1.58 6.02E-03 4.67E-02 SMG1 homolog, phosphatidylinositol 3-kinase-related kinase 23049 NM_015092 (C. elegans) VPS13D 1.59 1.05E-03 1.63E-02 vacuolar protein sorting 13 homolog D (S. cerevisiae) 55187 NM_015378

CPD 1.59 4.07E-03 3.65E-02 carboxypeptidase D 1362 NM_001304

CLIP1 1.59 1.19E-03 1.76E-02 CAP-GLY domain containing linker protein 1 6249 NM_002956

LAMP1 1.59 2.80E-04 8.57E-03 lysosomal-associated membrane protein 1 3916 NM_005561

PLD1 1.59 2.82E-05 3.19E-03 D1, phosphatidylcholine-specific 5337 NM_002662

ARHGDIB 1.60 6.28E-03 4.79E-02 Rho GDP dissociation inhibitor (GDI) beta 397 NM_001175

MAP3K7 1.60 4.44E-04 1.06E-02 mitogen-activated protein kinase kinase kinase 7 6885 NM_003188

DSC2 1.60 3.63E-03 3.42E-02 desmocollin 2 1824 NM_004949

GATM 1.60 5.03E-03 4.14E-02 glycine amidinotransferase (L-arginine:glycine 2628 NM_001482 amidinotransferase) SMAD5 1.60 5.26E-05 4.23E-03 SMAD family member 5 4090 NM_005903

PKIA 1.61 4.92E-04 1.11E-02 protein kinase (cAMP-dependent, catalytic) inhibitor alpha 5569 NM_006823

RASA2 1.61 7.05E-05 4.70E-03 RAS p21 protein activator 2 5922 NM_006506

UBL3 1.61 1.71E-03 2.19E-02 ubiquitin-like 3 5412 NM_007106

SLC38A7 1.61 4.12E-03 3.68E-02 solute carrier family 38, member 7 55238 NM_018231

KDM6B 1.62 3.29E-03 3.23E-02 lysine (K)-specific 6B 23135 NM_001080424

ESRRG 1.62 1.71E-05 2.56E-03 estrogen-related receptor gamma 2104 NM_001438

IDS 1.62 3.86E-03 3.54E-02 iduronate 2- 3423 NM_000202

PON1 1.62 9.21E-04 1.54E-02 paraoxonase 1 5444 NM_000446

ONECUT1 1.63 2.02E-03 2.42E-02 one cut homeobox 1 3175 NM_004498

KDM5B 1.63 5.56E-04 1.19E-02 lysine (K)-specific demethylase 5B 10765 NM_006618

MYO6 1.63 5.15E-03 4.20E-02 VI 4646 NM_004999

FAM155A 1.63 2.16E-04 7.49E-03 family with sequence similarity 155, member A 728215 NM_001080396

ZNF254 1.63 1.98E-04 7.25E-03 zinc finger protein 254 9534 NM_203282

GPR37 1.64 5.48E-03 4.38E-02 G protein-coupled receptor 37 (endothelin receptor type B- 2861 NM_005302 like) POU4F1 1.64 5.45E-03 4.37E-02 POU class 4 homeobox 1 5457 NM_006237

TCIRG1 1.65 1.14E-03 1.71E-02 T-cell, immune regulator 1, ATPase, H+ transporting, 10312 NM_006019 lysosomal V0 subunit A3 SSX4 1.65 1.78E-03 2.25E-02 synovial sarcoma, X breakpoint 4 /// synovial sarcoma, X 6759 NM_005636 breakpoint 4B RAB2A 1.65 5.96E-04 1.22E-02 RAB2A, member RAS oncogene family 5862 NM_002865

INHBE 1.65 4.98E-04 1.12E-02 inhibin, beta E 83729 NM_031479

JAK2 1.65 2.50E-03 2.74E-02 Janus kinase 2 3717 NM_004972

KRIT1 1.65 2.56E-03 2.77E-02 KRIT1, containing 889 NM_004912

SERPINI1 1.65 2.74E-05 3.17E-03 serpin peptidase inhibitor, clade I (neuroserpin), member 1 5274 NM_005025

INSIG1 1.66 1.93E-06 1.40E-03 insulin induced gene 1 3638 NM_005542

SP100 1.66 4.49E-03 3.88E-02 6672 NM_003113

RNF38 1.66 1.12E-04 5.74E-03 ring finger protein 38 152006 NM_022781

CNTNAP2 1.66 5.01E-03 4.13E-02 contactin associated protein-like 2 26047 NM_014141

SI 52

GLIPR1 1.67 2.35E-03 2.63E-02 GLI pathogenesis-related 1 11010 NM_006851

TMC7 1.67 4.07E-04 1.03E-02 transmembrane channel-like 7 79905 NM_024847

RPS11 1.68 4.81E-03 4.04E-02 Ribosomal protein S11 6205 NM_001015

CAV1 1.68 7.71E-04 1.41E-02 , caveolae protein, 22kDa 857 NM_001753

FNDC4 1.68 2.38E-03 2.64E-02 fibronectin type III domain containing 4 64838 NM_022823

CUL5 1.69 1.73E-04 6.92E-03 cullin 5 8065 NM_003478

DDX3X 1.69 1.17E-04 5.81E-03 DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked 1654 NM_001356

TBC1D9 1.70 6.15E-04 1.24E-02 TBC1 domain family, member 9 (with GRAM domain) 23158 NM_015130

KIAA1466 1.70 5.11E-05 4.20E-03 KIAA1466 gene 57612

WSB1 1.70 3.80E-03 3.51E-02 WD repeat and SOCS box-containing 1 26118 NM_015626

VCAN 1.70 2.48E-04 8.11E-03 versican 1462 NM_004385

TIPARP 1.71 3.05E-04 8.87E-03 TCDD-inducible poly(ADP-ribose) polymerase 25976 NM_015508

PHLDA1 1.72 1.57E-03 2.08E-02 pleckstrin homology-like domain, family A, member 1 22822 NM_007350

TXN 1.72 6.42E-04 1.28E-02 Thioredoxin 7295 NM_003329

JUN 1.72 1.37E-04 6.19E-03 jun proto-oncogene 3725 NM_002228

KIAA0247 1.73 7.75E-06 1.97E-03 KIAA0247 9766 NM_014734

LMAN1 1.73 1.85E-03 2.30E-02 lectin, mannose-binding, 1 3998 NM_005570

LRP12 1.73 3.20E-03 3.18E-02 low density lipoprotein receptor-related protein 12 29967 NM_013437

CCNG2 1.73 4.24E-03 3.75E-02 cyclin G2 901 NM_004354

NTAN1 1.73 5.09E-06 1.71E-03 N-terminal asparagine amidase 123803 NM_173474

ACVR1B 1.73 2.50E-03 2.74E-02 activin A receptor, type IB 91 NM_004302

SOS1 1.73 2.65E-04 8.35E-03 homolog 1 (Drosophila) 6654 NM_005633

SIK1 1.75 3.34E-04 9.27E-03 salt-inducible kinase 1 150094 NM_173354

MTMR9 1.75 1.84E-06 1.40E-03 myotubularin related protein 9 66036 NM_015458

HNRNPD 1.75 4.17E-04 1.04E-02 Heterogeneous nuclear ribonucleoprotein D (AU-rich element 3184 NM_002138 RNA binding protein 1, 37kDa) NPEPPS 1.78 9.98E-04 1.59E-02 Aminopeptidase puromycin sensitive 9520 NM_006310

IL8 1.78 4.05E-04 1.03E-02 interleukin 8 3576 NM_000584

TNFAIP3 1.78 2.73E-04 8.50E-03 tumor necrosis factor, alpha-induced protein 3 7128 NM_006290

FZD5 1.79 1.40E-04 6.24E-03 frizzled homolog 5 (Drosophila) 7855 NM_003468

NBPF1 1.79 1.62E-03 2.13E-02 neuroblastoma breakpoint family, member 1 55672 NM_017940

ANKRD5 1.80 1.25E-05 2.31E-03 ankyrin repeat domain 5 63926 NM_022096

PRKAB2 1.80 8.32E-04 1.46E-02 protein kinase, AMP-activated, beta 2 non-catalytic subunit 5565 NM_005399

TNFAIP8 1.81 5.99E-05 4.52E-03 tumor necrosis factor, alpha-induced protein 8 25816 NM_014350

FHOD3 1.81 9.60E-05 5.32E-03 formin homology 2 domain containing 3 80206 NM_025135

MED14 1.82 1.40E-03 1.94E-02 mediator complex subunit 14 9282 NM_004229

MR1 1.82 4.84E-03 4.04E-02 major histocompatibility complex, class I-related 3140 NM_001531

CADM1 1.82 7.39E-05 4.75E-03 cell adhesion molecule 1 23705 NM_014333

CPNE3 1.83 1.58E-03 2.09E-02 copine III 8895 NM_003909

PRSS23 1.83 3.95E-04 1.02E-02 protease, serine, 23 11098 NM_007173

ATF3 1.84 9.54E-04 1.55E-02 activating transcription factor 3 467 NM_001674

GADD45B 1.84 4.24E-03 3.75E-02 Growth arrest and DNA-damage-inducible, beta 4616 NM_015675

RARRES3 1.84 1.17E-03 1.73E-02 responder (tazarotene induced) 3 5920 NM_004585

C18orf1 1.84 2.89E-05 3.22E-03 chromosome 18 open reading frame 1 753 NM_004338

SI 53

KLHDC8A 1.84 3.57E-03 3.39E-02 kelch domain containing 8A 55220 NM_018203

ARFGEF1 1.85 5.55E-04 1.19E-02 ADP-ribosylation factor guanine nucleotide-exchange factor 10565 NM_006421 1(brefeldin A-inhibited) UGT2B15 1.88 6.07E-04 1.22E-02 UDP glucuronosyltransferase 2 family, polypeptide B15 7366 NM_001076

LTBP1 1.88 1.48E-03 2.01E-02 latent transforming growth factor beta binding protein 1 4052 NM_000627

DIAPH2 1.88 3.15E-04 9.06E-03 diaphanous homolog 2 (Drosophila) 1730 NM_006729

SERPINE1 1.88 2.16E-03 2.51E-02 serpin peptidase inhibitor, clade E (nexin, plasminogen 5054 NM_000602 activator inhibitor type 1), member 1 TRIM36 1.89 4.58E-04 1.09E-02 tripartite motif-containing 36 55521 NM_018700

KIAA1033 1.89 2.66E-04 8.35E-03 KIAA1033 23325 NM_015275

GIPC2 1.90 1.16E-04 5.79E-03 GIPC PDZ domain containing family, member 2 54810 NM_017655

PDGFA 1.91 1.61E-06 1.37E-03 platelet-derived growth factor alpha polypeptide 5154 NM_002607

ZBTB6 1.91 5.90E-05 4.52E-03 zinc finger and BTB domain containing 6 10773 NM_006626

SGK3 1.91 4.34E-04 1.06E-02 serum/glucocorticoid regulated kinase family, member 3 23678 NM_013257

WEE1 1.92 6.48E-04 1.29E-02 WEE1 homolog (S. pombe) 7465 NM_003390

TRIO 1.92 2.00E-05 2.69E-03 triple functional domain (PTPRF interacting) 7204 NM_007118

TGFA 1.94 2.38E-03 2.65E-02 transforming growth factor, alpha 7039 NM_003236

FLRT2 1.99 3.26E-04 9.21E-03 fibronectin leucine rich transmembrane protein 2 23768 NM_013231

NBPF10 2.01 1.00E-03 1.59E-02 neuroblastoma breakpoint family, member 10 100132406 NM_001039703

SIKE1 2.01 7.06E-06 1.88E-03 suppressor of IKBKE 1 80143 NM_025073

ACOX3 2.02 3.49E-05 3.62E-03 acyl-CoA oxidase 3, pristanoyl 8310 NM_003501

PICALM 2.03 9.03E-04 1.52E-02 phosphatidylinositol binding assembly protein 8301 NM_007166

AREG 2.04 1.13E-04 5.76E-03 amphiregulin 374 NM_001657

NCF2 2.05 4.62E-04 1.09E-02 neutrophil cytosolic factor 2 4688 NM_000433

TUSC3 2.05 7.04E-04 1.34E-02 tumor suppressor candidate 3 7991 NM_006765

APBA2 2.06 3.10E-03 3.12E-02 amyloid beta (A4) precursor protein-binding, family A, 321 NM_005503 member 2 SNAP25 2.06 7.38E-05 4.75E-03 synaptosomal-associated protein, 25kDa 6616 NM_003081

GBP1 2.06 1.19E-03 1.75E-02 guanylate binding protein 1, interferon-inducible, 67kDa 2633 NM_002053

FBN1 2.10 1.15E-04 5.76E-03 1 2200 NM_000138

GABRB1 2.14 4.36E-03 3.81E-02 gamma-aminobutyric acid (GABA) A receptor, beta 1 2560 NM_000812

PION 2.17 2.21E-03 2.55E-02 pigeon homolog (Drosophila) 54103 NM_017439

CXCL10 2.18 7.29E-04 1.37E-02 chemokine (C-X-C motif) ligand 10 3627 NM_001565

HDAC9 2.18 4.83E-04 1.10E-02 9 9734 NM_014707

IL1RAP 2.20 1.07E-06 1.30E-03 interleukin 1 receptor accessory protein 3556 NM_134470

RND1 2.20 4.70E-05 4.01E-03 Rho family GTPase 1 27289 NM_014470

ANGPTL2 2.20 4.05E-06 1.60E-03 angiopoietin-like 2 23452 NM_012098

SCYL2 2.27 1.59E-04 6.64E-03 SCY1-like 2 (S. cerevisiae) 55681 NM_017988

LDHB 2.38 2.39E-03 2.65E-02 lactate dehydrogenase B 3945 NM_002300

FZD2 2.50 3.19E-03 3.18E-02 frizzled homolog 2 (Drosophila) 2535 NM_001466

MAB21L2 2.52 1.51E-05 2.39E-03 mab-21-like 2 (C. elegans) 10586 NM_006439

PDE5A 2.61 3.03E-03 3.08E-02 phosphodiesterase 5A, cGMP-specific 8654 NM_001083

TPM4 2.62 1.06E-05 2.24E-03 4 7171 NM_003290

SP4 2.79 1.54E-04 6.54E-03 6671 NM_003112

THBS1 2.83 3.38E-03 3.28E-02 thrombospondin 1 7057 NM_003246

SC5DL 2.86 1.30E-04 6.05E-03 sterol-C5-desaturase (ERG3 delta-5-desaturase homolog, S. 6309 NM_006918 cerevisiae)-like

SI 54

PDGFB 3.02 1.88E-04 7.11E-03 platelet-derived growth factor beta polypeptide (simian 5155 NM_002608 sarcoma viral (v-sis) oncogene homolog) APPBP2 3.17 4.40E-03 3.82E-02 amyloid beta precursor protein (cytoplasmic tail) binding 10513 NM_006380 protein 2 OR1A2 3.55 3.10E-03 3.12E-02 olfactory receptor, family 1, subfamily A, member 2 26189 NM_012352

LEFTY2 3.58 5.14E-06 1.71E-03 left-right determination factor 2 7044 NM_003240

CYP1B1 3.63 4.11E-04 1.04E-02 cytochrome P450, family 1, subfamily B, polypeptide 1 1545 NM_000104

TNFAIP6 3.74 3.97E-03 3.60E-02 tumor necrosis factor, alpha-induced protein 6 7130 NM_007115

SRSF5 3.82 1.47E-03 2.00E-02 serine/arginine-rich splicing factor 5 6430 NM_006925

GAD1 4.10 3.22E-03 3.19E-02 glutamate decarboxylase 1 (brain, 67kDa) 2571 NM_000817

CAPN11 5.13 5.75E-03 4.52E-02 calpain 11 11131 NM_007058

HES1 5.42 5.02E-03 4.14E-02 hairy and enhancer of split 1, (Drosophila) 3280 NM_005524

PTHLH 5.89 2.03E-03 2.42E-02 parathyroid hormone-like hormone 5744 NM_002820

FGF12 6.03 2.53E-04 8.13E-03 fibroblast growth factor 12 2257 NM_004113

Table S3. Predicted (significant) altered transcription factor activity in miR-223 altered gene set. FC, fold change. Transcription factor (TF) altered, original gene in the miR-223 altered dataset; Transcription Factor, TF name in MetaBase; Actual, number of altered TF targets in the miR-223 dataset; n, number of altered genes/probes in the miR-223 altered dataset; R, number of TF targets in MetaCore database or background list; Expected, mean value for hypergeometric distribution (n*R/N); Ratio, connectivity ratio (Actual/Expected); z-score, z-score ((Actual-Expected)/sqrt(variance)); P-Value, probability to have the given value of Actual or higher (or lower for negative z-score) (MetaCore™ version 6.11 build 41105).

Altered TF z- TF Fold TF Corrected Targets n R Expected Ratio p-value TF (complex) score Change P-value (mRNA)

c-Myc 105 415 2446 40.33 2.6 1.88E-20 10.81 SP1 72 415 1512 24.93 2.89 3.28E-16 9.81 CREB1 156 415 5162 85.11 1.83 5.47E-16 8.69 EGR1 36 415 428 7.06 5.1 1.33E-15 11.08 GCR-alpha 61 415 1172 19.32 3.16 1.58E-15 9.79 RelA (p65 NF-kB subunit) 47 415 758 12.50 3.76 6.88E-15 9.99 45 415 874 14.41 3.12 1.63E-11 8.27 ETS1 28 415 371 6.12 4.58 2.82E-11 8.99 c-Fos 21 415 262 4.32 4.86 3.03E-09 8.13 HIF1A 32 415 581 9.58 3.34 3.24E-09 7.39 c-Jun 36 415 742 12.23 2.94 9.26E-09 6.95 JUN 1.72 0.006 RUNX2 15 415 146 2.41 6.23 2.09E-08 8.21 C/EBPbeta 30 415 575 9.48 3.16 3.43E-08 6.80 p53 42 415 1011 16.67 2.52 4.44E-08 6.39 TCF7L2 (TCF4) 22 415 334 5.51 4 4.55E-08 7.13 TCF7L2 -1.55 0.010

SI 55

FOXM1 12 415 98 1.62 7.43 7.80E-08 8.25 SMAD3 17 415 209 3.45 4.93 7.90E-08 7.39 STAT3 28 415 537 8.85 3.16 1.01E-07 6.56 p63 24 415 413 6.81 3.52 1.18E-07 6.70 Oct-3/4 45 415 1186 19.56 2.3 1.94E-07 5.94 ESR1 (nuclear) 45 415 1191 19.64 2.29 2.19E-07 5.91 CUX1 (p110) 50 415 1421 23.43 2.13 3.80E-07 5.70 WT1 13 415 138 2.28 5.71 5.06E-07 7.19 NF-kB1 (p50) 23 415 438 7.22 3.19 1.26E-06 5.97 MafK 5 415 13 0.21 23.3 1.37E-06 10.43 PPAR- gamma 17 415 257 4.24 4.01 1.47E-06 6.28 C/EBPalpha 20 415 349 5.75 3.48 1.67E-06 6.03 SP3 24 415 483 7.96 3.01 1.94E-06 5.79 FKHR 15 415 209 3.45 4.35 2.23E-06 6.30 FOXO1 -1.58 0.001 SRF 21 415 396 6.53 3.22 3.13E-06 5.76 RelB (NF-kB subunit) 14 415 196 3.23 4.33 5.13E-06 6.06 NANOG 29 415 702 11.57 2.51 6.57E-06 5.24 SMAD4 14 415 202 3.33 4.2 7.26E-06 5.92 AP-2A 16 415 262 4.32 3.7 8.25E-06 5.70 STAT1 18 415 326 5.38 3.35 9.16E-06 5.53 PR (nuclear) 15 415 236 3.89 3.86 9.81E-06 5.71 MITF 11 415 131 2.16 5.09 1.16E-05 6.08 VDR 13 415 183 3.02 4.31 1.18E-05 5.82 VDR -1.53 0.009 ESR2 11 415 132 2.18 5.05 1.25E-05 6.05 PU.1 19 415 373 6.15 3.09 1.62E-05 5.26 17 415 312 5.14 3.31 1.92E-05 5.30 Max 9 415 91 1.50 6 1.96E-05 6.19 JunB 10 415 117 1.93 5.18 2.48E-05 5.87 JUNB 1.55 0.039 29 415 756 12.47 2.33 2.60E-05 4.80 E2F1 -1.97 0.001 11 415 147 2.42 4.54 3.42E-05 5.57 JunD 12 415 177 2.92 4.11 4.03E-05 5.38 PPAR- beta(delta) 9 415 101 1.67 5.4 4.53E-05 5.74 Elk-3 4 415 14 0.23 17.3 6.40E-05 7.91 0.000103 ATF-3 8 415 88 1.45 5.51 9 5.49 ATF3 1.84 0.015 0.000115 13 415 228 3.76 3.46 3 4.83 NF-kB 0.000128 p50/p50 7 415 68 1.12 6.24 5 5.61 AHR 13 415 233 3.84 3.38 0.000143 4.73 0.000148 HSF1 14 415 266 4.39 3.19 2 4.65 DEC1 0.000154 (Stra13) 7 415 70 1.15 6.07 5 5.49 HNF4-alpha 19 415 444 7.32 2.6 0.000165 4.39 0.000166 KLF5 6 415 50 0.82 7.28 8 5.75 KLF5 -1.75 0.004 0.000170 C/EBPdelta 10 415 147 2.42 4.13 4 4.92 CDX2 8 415 99 1.63 4.9 0.000236 5.04 CDX2 -2.50 0.008

SI 56

5 0.000266 GATA-2 11 415 185 3.05 3.61 3 4.61 0.000271 DP2 2 415 2 0.03 60.7 2 10.92 0.000271 TAFII150 2 415 2 0.03 60.7 2 10.92 0.000271 TSC-22 2 415 2 0.03 60.7 2 10.92 ATF-2 10 415 156 2.57 3.89 0.000276 4.68 0.000283 FosB 6 415 55 0.91 6.62 9 5.40 0.000295 YY1 35 415 1132 18.67 1.88 1 3.90 0.000305 NRF2 10 415 158 2.61 3.84 7 4.63 0.000335 CDX1 5 415 37 0.61 8.2 8 5.67 0.000345 ARNT 6 415 57 0.94 6.38 7 5.27 0.000381 ZNF281 5 415 38 0.63 7.98 5 5.58 0.000381 PAX3 5 415 38 0.63 7.98 5 5.58 0.000415 Lef-1 9 415 135 2.23 4.04 1 4.59 LEF1 -1.59 0.005 0.000597 PPAR-alpha 10 415 172 2.84 3.53 6 4.30 c-Rel (NF-kB 0.000660 subunit) 11 415 206 3.40 3.24 3 4.18 0.000704 Fra-1 6 415 65 1.07 5.6 6 4.81 0.000804 NKRF 2 415 3 0.05 40.4 8 8.84 0.000828 MEF2A 6 415 67 1.11 5.43 2 4.70 9 415 149 2.46 3.66 0.000847 4.22 N-Myc 11 415 218 3.60 3.06 0.00105 3.96 TCF7 (TCF1) 4 415 28 0.46 8.66 0.00109 5.25 SNAIL1 6 415 71 1.17 5.13 0.001125 4.51 NRL 5 415 48 0.79 6.32 0.001137 4.78 CLOCK 6 415 73 1.20 4.99 0.001301 4.42 TCF7L1 (TCF3) 6 415 73 1.20 4.99 0.001301 4.42 PEA3 6 415 74 1.22 4.92 0.001396 4.37 RARalpha 8 415 130 2.14 3.73 0.001444 4.04 NF-E2 (45 kDa) 5 415 51 0.84 5.95 0.001499 4.58 SMAD2 8 415 132 2.18 3.68 0.001591 3.99 17 415 455 7.50 2.27 0.001601 3.53 MEF2D 4 415 31 0.51 7.83 0.001611 4.92 TAF1 4 415 31 0.51 7.83 0.001611 4.92 PAX5 8 415 136 2.24 3.57 0.001922 3.89 TFE3 4 415 33 0.54 7.35 0.002042 4.73 FOXC2 4 415 33 0.54 7.35 0.002042 4.73 TEF-4 3 415 16 0.26 11.4 0.002124 5.37 7 415 111 1.83 3.83 0.002462 3.86 Pitx1 3 415 17 0.28 10.7 0.002548 5.18 RBP-J kappa 7 415 112 1.85 3.79 0.00259 3.83 SI 57

(CBF1)

OLIG1 2 415 5 0.08 24.3 0.002624 6.74 FoxL2 5 415 58 0.96 5.23 0.002661 4.17 USF1 10 415 211 3.48 2.87 0.002762 3.54 HNF3-alpha 9 415 177 2.92 3.08 0.002781 3.60 ATF-4 6 415 85 1.40 4.28 0.002834 3.92 TR-alpha 6 415 87 1.44 4.18 0.003184 3.85 SMAD1 7 415 117 1.93 3.63 0.003307 3.69 Elk-1 8 415 149 2.46 3.26 0.003382 3.58 GATA-3 20 415 627 10.34 1.94 0.004076 3.07 POLR2A 3 415 21 0.35 8.66 0.004747 4.55 EPAS1 8 415 158 2.61 3.07 0.004816 3.38 HLF -3.35 0.015 NFYA 7 415 126 2.08 3.37 0.004968 3.45 Bcl-6 7 415 126 2.08 3.37 0.004968 3.45 DMTF1 2 415 7 0.12 17.3 0.005392 5.59 NRF3 2 415 7 0.12 17.3 0.005392 5.59 NRF1 7 415 128 2.11 3.32 0.005409 3.40 3 415 22 0.36 8.27 0.00543 4.42 NUR77 4 415 44 0.73 5.51 0.005878 3.88 TBP 6 415 99 1.63 3.68 0.005996 3.45 ITF2 3 415 23 0.38 7.91 0.006169 4.29 GLI-2 4 415 45 0.74 5.39 0.006369 3.82 SMAD5 5 415 72 1.19 4.21 0.006741 3.53 SMAD5 1.60 0.004 Basonuclin 2 415 8 0.13 15.2 0.007111 5.19 FoxD1 2 415 8 0.13 15.2 0.007111 5.19 FOXD1 -1.52 0.048 Bcl-3 2 415 8 0.13 15.2 0.007111 5.19 STAT5A 6 415 103 1.70 3.53 0.007245 3.34 SOX9 6 415 103 1.70 3.53 0.007245 3.34 BMAL1 6 415 104 1.72 3.5 0.007585 3.31 GATA-6 6 415 105 1.73 3.47 0.007936 3.28 CHD7 4 415 49 0.81 4.95 0.008604 3.58 SREBP1 (nuclear) 7 415 140 2.31 3.03 0.008702 3.12 NF-kB p65/p65 3 415 26 0.43 7 0.008732 3.96 FOXO3A 8 415 175 2.89 2.77 0.008743 3.05 TEF-1 4 415 51 0.84 4.76 0.009891 3.48 IRF8 9 415 220 3.63 2.48 0.01114 2.86 CREB3 2 415 10 0.16 12.1 0.01118 4.56 ETS2 6 415 114 1.88 3.19 0.01164 3.04 E2F6 5 415 84 1.39 3.61 0.01269 3.10 RFX1 3 415 30 0.49 6.07 0.01299 3.59 NOTCH1 (NICD) 2 415 11 0.18 11 0.01352 4.31 NRSF 13 415 391 6.45 2.02 0.01362 2.62 TITF1 5 415 87 1.44 3.49 0.01459 3.01 EBF 6 415 121 2.00 3.01 0.01525 2.87 0.53 ATH1 3 415 32 5.69 0.01549 3.43 0.20 ATF-5 2 415 12 10.1 0.01605 4.09

SI 58

TEF-3 4 415 59 0.97 4.11 0.01626 3.10 TFIIA 0.02 gamma chain 1 415 1 60.7 0.01649 7.72 MAD1 0.02 (mitotic checkpoint) 1 415 1 60.7 0.01649 7.72 MAD1L1 -1.81 0.012 PHF14 1 415 1 0.02 60.7 0.01649 7.72 HuD 0.02 (ELAVL-4) 1 415 1 60.7 0.01649 7.72 0.02 ZNF174 1 415 1 60.7 0.01649 7.72 BTBD14A 1 415 1 0.02 60.7 0.01649 7.72 IL-6 1 415 1 0.02 60.7 0.01649 7.72 PRR13 1 415 1 0.02 60.7 0.01649 7.72 1 415 1 0.02 60.7 0.01649 7.72 0.02 TIF1-gamma 1 415 1 60.7 0.01649 7.72 0.02 REXO4 1 415 1 60.7 0.01649 7.72 STK16 1 415 1 0.02 60.7 0.01649 7.72 ELL 1 415 1 0.02 60.7 0.01649 7.72 Ebp1 1 415 1 0.02 60.7 0.01649 7.72 HOXD13 3 415 33 0.54 5.51 0.01684 3.36 0.99 AP-4 4 415 60 4.04 0.0172 3.06 1.50 b-Myb 5 415 91 3.33 0.01742 2.89

Table S4. Significant differential hepatic mRNA expression in miR-223-/- mice. (Benjamini-Hochberg Corrected P-value < 0.05; >1.5 absolute fold change). Top 50 altered genes (up and down).

Gene Fold P-value Corrected Gene Title Entrez Symbol Change P-value Gene Ctse 70.59 1.94E-08 1.29E-04 cathepsin E 13034 Ren1 45.74 1.12E-06 2.14E-03 renin 1 structural 19701 Gdpd3 38.66 2.71E-06 3.27E-03 glycerophosphodiester phosphodiesterase 68616 domain containing 3 Aqp4 15.43 1.97E-04 1.35E-02 aquaporin 4 11829 Snhg11 9.43 2.61E-03 4.13E-02 small nucleolar RNA host gene 11 319317 Pstpip2 9.12 2.65E-04 1.49E-02 proline-serine-threonine phosphatase-interacting 19201 protein 2 5330411 8.89 1.18E-04 1.07E-02 RIKEN cDNA 5330411O13 gene 76673 O13Rik Eif2b5 8.66 2.08E-12 8.30E-08 Eukaryotic translation initiation factor 2B, subunit 224045 5 epsilon Igh-6 8.61 4.85E-04 1.89E-02 immunoglobulin heavy chain 6 (heavy chain of 16019 IgM) Chrna4 8.25 3.44E-03 4.71E-02 cholinergic receptor, nicotinic, alpha polypeptide 11438 4 Gm3893 7.03 1.76E-07 6.40E-04 predicted gene 3893 100042539 5930434 6.67 4.55E-04 1.85E-02 RIKEN cDNA 5930434B04 gene 381356 B04Rik Klhdc7a 6.42 3.32E-04 1.64E-02 kelch domain containing 7A 242721 Gpc1 5.91 9.93E-04 2.57E-02 glypican 1 14733 Malat1 5.76 2.79E-06 3.28E-03 metastasis associated lung adenocarcinoma 72289

SI 59

transcript 1 (non-coding RNA) Car3 5.51 4.62E-04 1.85E-02 carbonic anhydrase 3 12350 Fgf9 5.48 5.56E-04 2.00E-02 fibroblast growth factor 9 14180 Dntt 5.46 7.14E-04 2.24E-02 deoxynucleotidyltransferase, terminal 21673 Unc80 5.28 4.78E-04 1.88E-02 unc-80 homolog (C. elegans) 329178 Ntrk2 5.25 3.52E-04 1.68E-02 neurotrophic tyrosine kinase, receptor, type 2 18212 Cops5 5.23 7.88E-09 6.29E-05 COP9 (constitutive photomorphogenic) homolog, 26754 subunit 5 (Arabidopsis thaliana) Arhgap2 5.03 6.54E-05 8.27E-03 Rho GTPase activating protein 26 71302 6 AW0462 5.02 3.22E-07 9.90E-04 expressed sequence AW046200 100502619 00 Gabrb2 4.96 1.59E-03 3.22E-02 gamma-aminobutyric acid (GABA) A receptor, 14401 subunit beta 2 Sbsn 4.90 2.89E-03 4.33E-02 suprabasin 282619 Rrm2b 4.70 1.57E-04 1.23E-02 ribonucleotide reductase M2 B (TP53 inducible) 382985 Cd36 4.64 1.08E-04 1.04E-02 CD36 antigen 12491 Dct 4.56 1.45E-05 5.36E-03 dopachrome tautomerase 13190 Art3 4.42 1.59E-03 3.22E-02 ADP-ribosyltransferase 3 109979 Vldlr 4.29 7.06E-04 2.23E-02 very low density lipoprotein receptor 22359 LOC100 4.28 2.27E-03 3.84E-02 hypothetical LOC100504522 100504522 504522 4930423 4.24 1.56E-03 3.20E-02 RIKEN cDNA 4930423K06 gene 100502740 K06Rik 4933411 4.22 8.19E-04 2.35E-02 RIKEN cDNA 4933411K20 gene 66756 K20Rik AI32404 4.19 2.66E-04 1.49E-02 expressed sequence AI324046 380795 6 Cyp7a1 4.13 1.28E-03 2.89E-02 cytochrome P450, family 7, subfamily a, 13122 polypeptide 1 Pck2 3.99 1.04E-03 2.62E-02 phosphoenolpyruvate carboxykinase 2 74551 (mitochondrial) Fitm1 3.89 2.63E-03 4.15E-02 fat storage-inducing transmembrane protein 1 68680 Cenpp 3.83 3.67E-03 4.88E-02 centromere protein P 66336 D5Ertd5 3.79 4.28E-04 1.81E-02 DNA segment, Chr 5, ERATO Doi 521, 52350 21e expressed Pltp 3.77 4.30E-05 7.00E-03 phospholipid transfer protein 18830 Slc6a15 3.75 1.52E-03 3.17E-02 solute carrier family 6 (neurotransmitter 103098 transporter), member 15 Ncam2 3.73 2.59E-03 4.12E-02 neural cell adhesion molecule 2 17968 BC0055 3.58 3.21E-03 4.56E-02 cDNA sequence BC005512 192885 12 Wee1 3.46 2.98E-04 1.57E-02 WEE 1 homolog 1 (S. pombe) 22390 Pde10a 3.44 4.60E-04 1.85E-02 phosphodiesterase 10A 23984 Speer5- 3.44 4.73E-04 1.87E-02 spermatogenesis associated glutamate (E)-rich 70365 ps1 protein 5, pseudogene 1 1700029 3.35 5.08E-04 1.92E-02 RIKEN cDNA 1700029I01 gene 195531 I01Rik Gadd45 3.32 1.23E-03 2.84E-02 growth arrest and DNA-damage-inducible 45 13197 a alpha Ces2c 3.31 6.79E-04 2.18E-02 carboxylesterase 2C 234671 Rcan2 3.29 5.88E-05 7.92E-03 regulator of 2 53901

SI 60

C03001 -4.48 1.12E-04 1.05E-02 RIKEN cDNA C030018P15 gene 109260 8P15Rik Mios -4.55 4.53E-04 1.85E-02 missing oocyte, meiosis regulator, homolog 252875 (Drosophila) Dock1 -4.58 1.24E-03 2.85E-02 dedicator of cytokinesis 1 330662 Ntng2 -4.64 3.70E-03 4.90E-02 netrin G2 171171 P4htm -4.80 1.53E-03 3.18E-02 prolyl 4-hydroxylase, transmembrane 74443 () Osbpl6 -4.80 6.79E-04 2.18E-02 oxysterol binding protein-like 6 99031 Svs5 -4.82 3.48E-04 1.67E-02 seminal vesicle secretory protein 5 20944 Gm3579 -4.83 1.21E-03 2.83E-02 predicted gene 3579 100041932 Cd44 -4.88 2.78E-03 4.24E-02 CD44 antigen 12505 Rbfox1 -4.98 1.25E-03 2.85E-02 RNA binding protein, fox-1 homolog (C. elegans) 268859 1 Sema5a -5.04 1.61E-03 3.24E-02 sema domain, seven thrombospondin repeats 20356 (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5A Ttll9 -5.15 2.31E-03 3.87E-02 tubulin tyrosine -like family, member 9 74711 Cd70 -5.22 2.20E-03 3.77E-02 CD70 antigen 21948 2410089 -5.24 2.39E-03 3.96E-02 RIKEN cDNA 2410089E03 gene 73692 E03Rik Elovl4 -5.25 2.51E-03 4.06E-02 elongation of very long chain fatty acids (FEN1 83603 Atp1a2 -5.25 1.18E-04 1.07E-02 ATPase, Na+ 98660 Ikbkb -5.26 2.96E-03 4.37E-02 inhibitor of kappaB kinase beta 16150 Pappa -5.28 9.63E-04 2.53E-02 pregnancy-associated plasma protein A 18491 Dmc1 -5.28 1.79E-03 3.42E-02 DMC1 dosage suppressor of mck1 homolog, 13404 meiosis-specific homologous recombination (yeast) Tlr7 -5.31 9.20E-05 9.47E-03 toll-like receptor 7 170743 4933402 -5.36 3.71E-03 4.90E-02 RIKEN cDNA 4933402C05 gene 71025 C05Rik C79657 -5.46 6.97E-04 2.21E-02 expressed sequence C79657 96987 Maml1 -5.49 2.13E-03 3.71E-02 Mastermind like 1 (Drosophila) 103806 Sh2d5 -5.53 1.87E-04 1.32E-02 SH2 domain containing 5 230863 Smarca -5.53 2.76E-05 5.96E-03 SWI 93762 5 4933422 -5.59 5.54E-04 2.00E-02 RIKEN cDNA 4933422A05 gene 71133 A05Rik Kcnj16 -5.70 6.72E-04 2.17E-02 potassium inwardly-rectifying channel, subfamily 16517 J, member 16 Tbc1d1 -5.85 2.27E-03 3.84E-02 TBC1 domain family, member 1 57915 Pcdh12 -5.85 9.15E-04 2.47E-02 protocadherin 12 53601 Arpp21 -5.92 3.46E-03 4.72E-02 cyclic AMP-regulated phosphoprotein, 21 74100 Rarg -6.31 1.00E-03 2.57E-02 retinoic acid receptor, gamma 19411 4930448 -6.72 2.62E-04 1.48E-02 RIKEN cDNA 4930448F12 gene 73992 F12Rik LOC640 -6.75 3.02E-03 4.42E-02 UDP-N-acetylhexosamine pyrophosphorylase- 640502 502 like 6430520 -7.07 1.03E-03 2.61E-02 RIKEN cDNA 6430520M22 gene 319716 M22Rik Lepr -7.15 4.53E-04 1.85E-02 leptin receptor 16847

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Cct3 -7.24 2.63E-03 4.15E-02 chaperonin containing Tcp1, subunit 3 (gamma) 12462 Tmem1 -7.31 3.60E-04 1.70E-02 transmembrane protein 107 66910 07 Stxbp5l -7.34 3.23E-03 4.57E-02 syntaxin binding protein 5-like 207227 Dgkb -7.92 4.57E-04 1.85E-02 diacylglycerol kinase, beta 217480 Raly -8.21 6.67E-04 2.17E-02 hnRNP-associated with lethal yellow 19383 Spdyb -8.36 7.73E-05 8.94E-03 speedy homolog B (Xenopus laevis) 74673 Arih1 -8.90 5.36E-04 1.97E-02 Ariadne ubiquitin-conjugating enzyme E2 binding 23806 protein homolog 1 (Drosophila) Prrx1 -10.42 1.58E-04 1.24E-02 paired related homeobox 1 18933 Cx3cl1 -11.86 2.31E-05 5.96E-03 chemokine (C-X3-C motif) ligand 1 20312 Mycn -12.09 2.04E-03 3.65E-02 v-myc myelocytomatosis viral related oncogene, 18109 neuroblastoma derived (avian) Mid1 -12.36 3.61E-05 6.39E-03 midline 1 17318 1700040 -13.65 1.46E-07 5.81E-04 RIKEN cDNA 1700040D17 gene 76602 D17Rik Zfp42 -14.59 3.75E-10 4.98E-06 zinc finger protein 42 22702 Sash3 -26.75 1.06E-03 2.64E-02 SAM and SH3 domain containing 3 74131 7120451 -41.12 2.99E-10 4.98E-06 RIKEN cDNA 7120451J01 gene 100038434 J01Rik

Table S5. Significantly up-regulated hepatic mRNAs expression in miR-223-/- mice that are predicted mouse miR-223 target genes (Benjamini-Hochberg Corrected P-value < 0.05; >1.5 absolute fold change).

Gene Fold P-value Corrected Gene Title Entrez Symbol Change P-value Gene Aqp4 15.43 1.35E-02 1.97E-04 aquaporin 4 11829 Pstpip2 9.12 1.49E-02 2.65E-04 proline-serine-threonine phosphatase- 19201 interacting protein 2 Ntrk2 5.25 1.68E-02 3.52E-04 neurotrophic tyrosine kinase, receptor, 18212 type 2 Arhgap26 5.03 8.27E-03 6.54E-05 Rho GTPase activating protein 26 71302 Pde10a 3.44 1.85E-02 4.60E-04 phosphodiesterase 10A 23984 Rcan2 3.29 7.92E-03 5.88E-05 regulator of calcineurin 2 53901 Ehbp1l1 2.71 1.97E-02 5.34E-04 EH domain binding protein 1-like 1 114601 Elovl6 2.59 3.71E-02 2.14E-03 ELOVL family member 6, elongation of 170439 long chain fatty acids (yeast) Mfsd9 2.54 4.76E-02 3.50E-03 major facilitator superfamily domain 211798 containing 9 Hmgcs1 2.54 1.47E-02 2.55E-04 3-hydroxy-3-methylglutaryl-Coenzyme A 208715 synthase 1 Pign 2.29 2.11E-02 6.26E-04 phosphatidylinositol glycan anchor 27392 biosynthesis, class N Trim37 2.27 5.36E-03 1.48E-05 tripartite motif-containing 37 68729 Prss23 2.26 1.97E-02 5.40E-04 protease, serine, 23 76453 F3 2.24 4.68E-02 3.37E-03 coagulation factor III 14066 Mpp5 2.22 9.13E-03 8.26E-05 membrane protein, palmitoylated 5 56217

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(MAGUK p55 subfamily member 5) Gna13 2.18 2.07E-03 1.04E-06 guanine nucleotide binding protein, alpha 14674 13 Tceal8 2.11 1.44E-02 2.38E-04 transcription A (SII)-like 66684 8 Tmem181a 2.05 3.12E-02 1.48E-03 transmembrane protein 181A /// 1E+08 transmembrane protein 181B, pseudogene /// transmembrane protein 181C, pseudogene /// transmembrane protein 181D, pseudogene Tcfcp2l1 2.03 1.79E-02 4.10E-04 transcription factor CP2-like 1 81879 Tlr3 2.02 4.13E-02 2.60E-03 toll-like receptor 3 142980 Serpinb8 2.01 3.16E-02 1.51E-03 serine (or cysteine) peptidase inhibitor, 20725 clade B, member 8 Rassf6 1.99 6.28E-03 3.23E-05 Ras association (RalGDS/AF-6) domain 73246 family member 6 Fam126a 1.96 2.48E-02 9.22E-04 family with sequence similarity 126, 84652 member A Pard6b 1.92 1.96E-02 5.30E-04 par-6 (partitioning defective 6) homolog 58220 beta (C. elegans) Fzd8 1.92 1.52E-02 2.75E-04 frizzled homolog 8 (Drosophila) 14370 Mras 1.90 4.25E-02 2.79E-03 muscle and microspikes RAS 17532 Rassf4 1.89 3.02E-02 1.40E-03 Ras association (RalGDS/AF-6) domain 213391 family member 4 Fam164a 1.89 3.65E-02 2.04E-03 family with sequence similarity 164, 67306 member A Cry2 1.88 1.59E-02 3.09E-04 cryptochrome 2 (photolyase-like) 12953 Acpp 1.88 4.95E-02 3.77E-03 , prostate 56318 Nqo1 1.86 2.01E-02 5.61E-04 NAD(P)H dehydrogenase, quinone 1 18104 Cyb5b 1.83 5.36E-03 1.49E-05 cytochrome b5 type B 66427 Entpd5 1.83 8.20E-03 6.25E-05 ectonucleoside triphosphate 12499 diphosphohydrolase 5 Igfbp5 1.81 3.77E-02 2.21E-03 insulin-like growth factor binding protein 16011 5 Kit 1.81 2.63E-02 1.05E-03 kit oncogene 16590 Nfkbie 1.80 1.82E-02 4.35E-04 nuclear factor of kappa light polypeptide 18037 gene enhancer in B-cells inhibitor, epsilon Vopp1 1.80 4.16E-02 2.64E-03 vesicular, overexpressed in cancer, 232023 prosurvival protein 1 Pgrmc2 1.80 1.64E-02 3.32E-04 membrane 70804 component 2 Ak3 1.75 1.42E-02 2.29E-04 adenylate kinase 3 56248 Lrrc8d 1.75 4.95E-02 3.77E-03 leucine rich repeat containing 8D 231549 Klhdc5 1.74 2.11E-02 6.28E-04 kelch domain containing 5 232539 Tex261 1.73 4.88E-03 1.04E-05 testis expressed gene 261 21766 Mapk9 1.73 7.37E-03 5.02E-05 mitogen-activated protein kinase 9 26420

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Ipo9 1.73 1.79E-02 4.11E-04 importin 9 226432 Rasa1 1.73 4.07E-02 2.52E-03 RAS p21 protein activator 1 218397 Prdx6 1.70 1.42E-02 2.29E-04 peroxiredoxin 6 11758 Psmf1 1.69 2.77E-02 1.16E-03 proteasome (prosome, macropain) 228769 inhibitor subunit 1 Cml2 1.67 3.03E-02 1.41E-03 camello-like 2 93673 D3Ertd254e 1.66 4.49E-02 3.09E-03 DNA segment, Chr 3, ERATO Doi 254, 241944 expressed Dock8 1.65 6.54E-03 3.77E-05 dedicator of cytokinesis 8 76088 Ido2 1.65 7.72E-03 5.56E-05 indoleamine 2,3-dioxygenase 2 209176 Kdelc2 1.65 2.02E-02 5.69E-04 KDEL (Lys-Asp-Glu-Leu) containing 2 68304 Zeb2 1.64 1.45E-02 2.44E-04 zinc finger E-box binding homeobox 2 24136 Cybb 1.63 4.38E-02 2.97E-03 cytochrome b-245, beta polypeptide 13058 Dcaf7 1.62 5.96E-03 2.56E-05 DDB1 and CUL4 associated factor 7 71833 Kif13a 1.62 4.20E-02 2.71E-03 Kinesin family member 13A 16553 Homer2 1.62 4.58E-02 3.25E-03 homer homolog 2 (Drosophila) 26557 Gpam 1.61 2.02E-02 5.71E-04 glycerol-3-phosphate acyltransferase, 14732 mitochondrial Prlr 1.60 2.85E-02 1.24E-03 prolactin receptor 19116 Prnp 1.59 6.15E-03 2.93E-05 prion protein 19122 Ets1 1.59 1.90E-02 4.96E-04 E26 avian leukemia oncogene 1, 5' 23871 domain Rbm47 1.58 7.37E-03 4.95E-05 RNA binding motif protein 47 245945 Tmem41b 1.57 5.28E-03 1.42E-05 transmembrane protein 41B 233724 Dbt 1.54 2.34E-02 8.07E-04 dihydrolipoamide branched chain 13171 transacylase E2 Sdf4 1.54 2.98E-02 1.36E-03 stromal cell derived factor 4 20318 Ttc28 1.54 2.90E-02 1.29E-03 tetratricopeptide repeat domain 28 209683 AI987944 1.53 1.74E-02 3.87E-04 expressed sequence AI987944 233168 Plekha8 1.52 4.26E-02 2.81E-03 pleckstrin homology domain containing, 231999 family A (phosphoinositide binding specific) member 8 Fign 1.52 4.56E-02 3.22E-03 fidgetin 60344 Sh3rf1 1.51 4.76E-02 3.51E-03 SH3 domain containing ring finger 1 59009 Slc35a5 1.51 2.11E-02 6.25E-04 solute carrier family 35, member A5 74102 Scaper 1.50 3.43E-02 1.81E-03 S phase cyclin A-associated protein in the 244891 ER

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Table S6. Significantly up-regulated hepatic mRNAs expression in miR-223-/- mice that are predicted mouse and human miR-223 target genes. (Benjamini- Hochberg Corrected P-value < 0.05; >1.5 absolute fold change).

Gene Fold P-value Corrected Gene Title Entrez Symbol Change P-value Gene Ntrk2 5.25 1.68E-02 3.52E-04 neurotrophic tyrosine kinase, receptor, type 2 18212 Hmgcs1 2.54 1.47E-02 2.55E-04 3-hydroxy-3-methylglutaryl-Coenzyme A 208715 synthase 1 Pign 2.29 2.11E-02 6.26E-04 phosphatidylinositol glycan anchor biosynthesis, 27392 class N Trim37 2.27 5.36E-03 1.48E-05 tripartite motif-containing 37 68729 F3 2.24 4.68E-02 3.37E-03 coagulation factor III 14066 Mpp5 2.22 9.13E-03 8.26E-05 membrane protein, palmitoylated 5 (MAGUK 56217 p55 subfamily member 5) Gna13 2.18 2.07E-03 1.04E-06 guanine nucleotide binding protein, alpha 13 14674 Serpinb8 2.01 3.16E-02 1.51E-03 serine (or cysteine) peptidase inhibitor, clade B, 20725 member 8 Rassf6 1.99 6.28E-03 3.23E-05 Ras association (RalGDS/AF-6) domain family 73246 member 6 Pard6b 1.92 1.96E-02 5.30E-04 par-6 (partitioning defective 6) homolog beta (C. 58220 elegans) Mras 1.90 4.25E-02 2.79E-03 muscle and microspikes RAS 17532 Cry2 1.88 1.59E-02 3.09E-04 cryptochrome 2 (photolyase-like) 12953 Acpp 1.88 4.95E-02 3.77E-03 acid phosphatase, prostate 56318 Entpd5 1.83 8.20E-03 6.25E-05 ectonucleoside triphosphate 12499 diphosphohydrolase 5 Klhdc5 1.74 2.11E-02 6.28E-04 kelch domain containing 5 232539 Ipo9 1.73 1.79E-02 4.11E-04 importin 9 226432 Rasa1 1.73 4.07E-02 2.52E-03 RAS p21 protein activator 1 218397 Zeb2 1.64 1.45E-02 2.44E-04 zinc finger E-box binding homeobox 2 24136 Dcaf7 1.62 5.96E-03 2.56E-05 DDB1 and CUL4 associated factor 7 71833 Prlr 1.60 2.85E-02 1.24E-03 prolactin receptor 19116 Dbt 1.54 2.34E-02 8.07E-04 dihydrolipoamide branched chain transacylase 13171 E2 Ttc28 1.54 2.90E-02 1.29E-03 tetratricopeptide repeat domain 28 209683 Sh3rf1 1.51 4.76E-02 3.51E-03 SH3 domain containing ring finger 1 59009

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Table S7. Significantly altered hepatic genes in miR-223-/- that have previously been associated with cholesterol and lipid homeostasis.(6-8)

Gene GWAS SREBP Symbol Lipid Targets Traits GPAM X X HMGCR X X LDLR X X MVK X X PCSK9 X X CAPN3 X CTF1 X CYP7A1 X JMJD1C X LRP4 X PLTP X ACSL4 X DHCR7 X ECHDC1 X ELOVL6 X FDFT1 X FDPS X HMGCS1 X LSS X MVD X NSDHL X SC4MOL X SQLE X STARD4 X

Supplemental References

1. Folch J, Lees M, & Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226(1):497-509. 2. Vickers KC, et al. (2013) MicroRNA-27b is a regulatory hub in lipid metabolism and is altered in dyslipidemia. Hepatology 57(2):533-542. 3. Li C HWW (2001) Model-based analysis of oligonucleotide arrays: model validation, design issues and standard error application. Genome Biol 2:RESEARCH0032.0031- 0032. 4. Foger B, et al. (1997) Plasma phospholipid transfer protein. Adenovirus-mediated overexpression in mice leads to decreased plasma high density lipoprotein (HDL) and

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enhanced hepatic uptake of phospholipids and cholesteryl esters from HDL. J. Biol. Chem. 272(43):27393-27400. 5. Vickers KC, Palmisano BT, Shoucri BM, Shamburek RD, & Remaley AT (2011) are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol 13(4):423-433. 6. Teslovich TM, et al. (2010) Biological, clinical and population relevance of 95 loci for blood lipids. Nature 466(7307):707-713. 7. Horton JD, et al. (2003) Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies direct SREBP target genes. Proc Natl Acad Sci U S A 100(21):12027-12032. 8. Seo YK, et al. (2011) Genome-wide localization of SREBP-2 in hepatic chromatin predicts a role in autophagy. Cell Metab 13(4):367-375.

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