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Supplementary Figure 1 A B mir-141, human cell lines mir-2c, human cell lines mir-141, hepatocytes mir-2c, hepatocytes Relative RNA.1.8.6.4.2 Relative RNA.3.2.1 Relative RNA 1.5 1..5 Relative RNA 2. 1.5 1..5. HepG2 Huh7. HepG2 Huh7.. WT mir-141/2c -/- WT mir-141/2c -/- Supplementary Fig. 1. qpcr analysis of mir-141 and mir-2c RNA expression levels (A) detected in two human hepatocellular carcinoma cell lines (HepG2 and Huh7) and (B) in primary mouse hepatocytes isolated from WT and mir-141/2c -/- mice (n=3 per group). Data are represented as mean ± SEM. A Students unpaired t-test was used to determine differences between WT and mir- 141/2c -/- mice. P<.5 and P<.1 indicate statistical significance.

Supplementary Figure 2 A B Serum Glucose (mg/dl) 12 8 4 CD MCD WT-CD mir-141/2c -/- -CD mir-141/2c -/- -MCD Cholesterol (µg/ml) 2. 1.5 1..5. mir-141/2c -/- -MCD MCD Supplementary Fig. 2A. Serum glucose levels in WT vs mir-141/2c -/- mice by calorimetric analysis (n=4-6 per group). Data are represented as mean ± SEM. P<.5 indicate statistical significance, one-way ANOVA with Newman-Keuls multiple comparisons test. Supplementary Fig. 2B. Liver cholesterol levels in vs mir-141/2c -/- -MCD livers by GC-TOF mass spectrometry (n=5 per group). Data are represented as mean ± SEM. C Oil Red O MCD Oil Red O MCD 2 µm WT mir-141/2c -/- 2 µm Oil Red O (relative surface area) 1.5 1..5. WT mir-141/2c -/- Supplementary Fig. 2C. Representative image of Oil red O staining showed reduced neutral lipid staining in vs mir-141/2c -/- -MCD livers. Images were quantified by digital image analysis using ImageJ software in 5 randomly chosen fields from 3 individual mice per group. Data are represented as mean ± SEM. Differences between WT and mir-141/2c -/- mice were compared using a Students unpaired t-test and P<.5 indicate statistical significance.

Supplementary Figure 3 A D Serum TBARS (µm) Relative mrna 6 4 2 Supplementary Fig. 3A. TBARS expression in serum and livers of vs mir- 141/2c -/- -MCD (n=5 per group). Data are represented as mean ± SEM. 2. 1.5 1..5 mir-141/2c -/- -MCD MCD Liver TBARS (mg/g) 1 8 6 4 2 mir-141/2c -/- -MCD P=.6 MCD B Amplex Red H 2 O 2 Fluorescence (AU) E 1 8 6 4 2 p-stat3 STAT3 mir-141/2c -/- -MCD MCD Supplementary Fig. 3B. Liver H 2 O 2 levels in vs mir-141/2c -/- -MCD (n=5-6 per group). Data are represented as mean ± SEM. WT KO 1..98 1..92 C Relative mrna 1 8 6 4 2 MMP2 ICAM α-sma Supplementary Fig. 3C. qpcr of fibrosis-related gene expression in vs mir-141/2c -/- -MCD livers (n=4-5 per group). Data are represented as mean ± SEM. P<.5 and P<.1 indicate statistical significance, one-way ANOVA with Newman-Keuls multiple comparisons test. WT-CD mir-141/2c -/- -CD mir-141/2c -/- -MCD. C/EBPβ STAT3 HIF1α α-tubulin Supplementary Fig. 3D. qpcr of gene expression in vs mir- 141/2c -/- -MCD livers (n=5 per group). Data are represented as mean ± SEM. Supplementary Fig. 3E. Western blot analysis of p-stat3 and STAT3 in WT- MCD vs mir-141/2c -/- -MCD livers. Densitometry of each blots relative to the loading control were quantified using ImageJ software. Samples were pooled from 5 individual mice per group.

Supplementary Figure 4 A CASPASE 3 CLEAVED CASPASE 3 β-actin WT KO 1..8 37 kda 25 kda 2 kda 15 kda B TUNEL-MCD WT mir-141/2c -/- 1 µm 1 µm TUNEL Positive Cells/Field 2 15 1 5 WT mir-141/2c -/- Supplementary Fig. 4A. Western blot analysis for apoptosis marker, caspase-3, in vs mir-141/2c -/- -MCD livers Densitometry of the blot relative to the loading control was quantified using ImageJ software. Samples were pooled from 5 individual mice per group. Supplementary Fig. 4B. Representative images of TUNEL assay for apoptosis in vs mir-141/2c -/- -MCD livers. The total number of TUNEL positive cells were counted across 1 random high resolution fields and were expressed as average number of TUNEL positive cells per field (n=1 per group). Data are represented as mean ± SEM.

Supplementary Figure 5 A TG (6:4) TG (6:3) TG (6:2) TG (6:12) TG (58:8) TG (58:6) TG (58:6) TG (58:4) A TG (58:3) TG (58:2) TG (58:1) TG (58:1) TG (56:9) TG (56:6) B TG (56:6) A TG (56:5) B TG (56:5) A TG (56:4) TG (56:3) TG (56:2) TG (54:8) TG (54:7) B TG (54:7) A TG (54:6) B TG (54:5) B TG (54:4) B TG (54:4) A TG (54:3) TG (54:2) TG (54:1) TG (53:5) TG (53:4) TG (53:3) TG (53:2) TG (52:6) B TG (52:6) A TG (52:6) TG (52:5) TG (52:4) TG (52:3) TG (52:2) TG (52:1) TG (51:4) TG (51:3) TG (51:2) TG (5:4) TG (5:2) TG (5:2) TG (5:1) TG (49:2) TG (48:3) TG (48:2) TG (48:1) WT mir-141/2c -/- 5x1 5 1x1 6 1.5x1 6 2x1 6 Average ion peak height B Supplementary Fig. 5. Reduced TGs in mir-141/2c -/- mice fed the MCD diet. Livers of WT and mir-141/2c -/- mice were prepared and subjected to LC-MS analysis for triglyceride (TG) lipid species (n=5 mice per group). (A) TG lipid species are presented as average ion peak height in order of increasing chain length (top to bottom). (B) Correlation matrix showing correlation coefficients for TG lipid species with Pearson r used for the distance measure. Positive correlations are displayed in red and negative correlations in blue color. Color intensity are proportional to the correlation coefficients. Correlated metabolites were highlighted with red box.

Supplementary Figure 6 A 5 mir-141/2c -/- -MCD 4 3 2 1 FA (16:1) FA (18:1) FA (18:2) FA (18:3) FA (2:1) FA (2:2) FA (2:3) FA (2:3) FA (2:4) FA (2:5) FA (22:) FA (22:6) FA (24:1) B PE (34:1) PE (34:2) PE (36:2) PE (36:3) PE (36:4) PE (38:5) PE (38:6) PE (4:5) PE (4:6) PE (4:7) PE (p-36:4) or PE (o-36:5) PE (p-38:4) or PE (o-38:5) PE (p-38:5) or PE (o-38:6) PE (p-38:6) or PE (o-38:7) PE (p-4:4) or PE (o-4:5) Relative Phosphatidyl Ethanoalamine Species Relative Fatty Acid Species 6 4 2 Supplementary Fig. 6. Lipidomics analysis revealed distinct changes in a number of lipid species associated with mir-141/2c. Livers of WT and mir-141/2c -/- mice were prepared and subjected to LC-MS for lipidomics analysis of various lipid classes (n=5 mice per group) including (A) fatty acids and (C) phosphatidylethanolamine (PE) lipid species and are expressed as fold change relative to WT mice. Data are represented as mean ± SEM. P <.5 versus WT by Students unpaired t-test.

Supplementary Figure 7 Supplementary Fig. 7. Correlation matrix showing distinct changes in amino acids from metabolomics analysis. Livers of WT and mir-141/2c -/- mice were prepared and subjected to LC-MS for metabolomics analysis (n=5 mice per group). Correlation matrix showing correlation coefficients with Pearson r used for the distance measure. Positive correlations are displayed in red and negative correlations in blue color. Color intensity are proportional to the correlation coefficients. Correlated metabolites were highlighted with red box.

Supplementary Figure 8 isohexonic acid -log1(p) 6 6 4 4 2 2 ornithine myristic acid histidine aminomalonate glyceric acid cholic acid myo-inositol lauric acid glycerol heptadecanoic acid 2 2 4 4 6 6 2 4 6 8 1 12 Compounds Supplementary Fig. 8. Metabolomics analysis revealed significantly altered metabolites associated with mir-141/2c-deficiency. Livers of WT and mir-141/2c -/- mice were prepared and subjected to LC-MS for metabolomics analysis (n=5 mice per group). A Students t- test showing metabolites that are significantly altered between WT and mir-141/2c -/- mice. Each dot represents a metabolite plotted as a compound number (x-axis) and statistical significance ( log 1 (p-value), y-axis).

SUPPLEMENTAL INFORMATION Loss of mir-141/2c Ameliorates Hepatic Steatosis and Inflammation by Reprograming Multiple Signaling Pathways in NASH Melanie Tran, Sang-Min Lee, Dong-Ju Shin, and Li Wang Table of Contents Supplementary Table 1... 2 Supplementary Table 2... 4 1

Supplementary Table 1. Sequences of primers used for quantitative-pcr Gene Forward Primer Sequence (5 3 ) Reverse Primer Sequence (3 5 ) mir-2c antisense mir-141 antisense mir-2c stem loop mir-141 stem loop Universal CDCTAATACTGCCGGGTAATG GCCCGCTAACACTGTCTGGTAAAG GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCATCA GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCATCT GTGCAGGGTCCGAGGT U6 CTCGCTTCGGCAGCACA AACGCTTCACGAATTTGCGT HPRT CACAGGACTAGAACACCTGC GCTGGTGAAAAGGACCTCT SREBP1c TTGCTGGCTTGGTGATGCTATG CTGGTGGAGGGCTGGAAGG FAS TCGGGTGTGGTGGGTTTGG GCGTGAGATGTGTTGCTGAGG MTTP CATGCTTCTTCATCTGGTCCG CACTTTGTCTTGCTGGGCCG SCD1 AGTTCCGCCACTCGCCTAC GATAGTCAGTTGCTCGCCTCAC HMGCR CCAAGCCCAATGAAGGGAAAGTC CCACAGGAACAAGGCACACAG LDLR GTGAGGTTCCTGTCCATCTTCTTC GTTCTTCAGCCGCCAGTTCC CYP7A1 CACATCCTCCTGGCATTTACCC TCCTCCTGTCTATGTCACACTACC SAA1 TGATGGAAGAGAGGCCTTTCA TCAGGCAGTCCAGGAGGTCT IL-1β CCTGTGTTTTCCTCCTTGCCT GCCTAATGTCCCCTTGAATCAA TNFα GACCCTCACACTCAGATCATC CCTCCACTTGGTGGTTTGCT IL-6 TGATGCACTTGCAGAAAACA ACCAGAGGAAATTTTCAATAGGC IL-4 TGAACGAGGTCACAGGAGAA CGAGCTCACTCTCTGTGGTG IL-1 ATCGATTTCTCCCCTGTGAA TGTCAAATTCATTCATGGCCT F4/8 CCCCAGTGTCCTTACAGAGTG GTGCCCAGAGTGGATGTCT LYG6 GACTTCCTGCAACACAACTAC ACAGCATTACCAGTGATCTCA 2

COL1A1 GCAGGGTTCCAACGATGTTG GCAGCCATCGACTAGGACAGA TGFβ GGAGACCCCTGGATACCAAC CAACCCAGGTCCTTCCTAAA α-sma AGAGTTACGAGTTGCCTGATG ATGAAGGATGGCTGGAACAG inos AATCTTGGAGCGAGTTGTGG CAGGAAGTAGGTGAGGGCTTG ARG1 TCCAAGCCAAAGTCCTTAGAG AGGAGCTGTCATTAGGGACATC C/EBPβ CAACCTGGAGACGCAGCACAA GGCAGCTGCTTGAACAAGTTC HIF1α GGGTACAAGAAACCACCCAT GAGGCTGTGTCGACTGAGAA STAT3 CAGAAAGTGTCCTACAAGGGCG CGTTGTTAGACTCCTCCATGTTC KLF4 GTGCCCCGACTAACCGTTG GTCGTTGAACTCCTCGGTCT IFR3 GGCTTGTGATGGTCAAGGTT CATGTCCTCCACCAAGTCCT ICAM GTCTCGGAAGGGAGCCAAGTA CGACGCCGCTCAGAAGAA MMP2 ATGCGGAAGCCAAGATGTG TTTCAGGGTCCAGGTCAGG NFκβ ATGATCCCTACGGAACTGGGCAAA TGGGCCATCTGTTGACAGTGGTAT 3

Supplementary Table 2. Clinical and biochemical characteristics of patients with non-alcoholic steatohepatitis (NASH) non fatty or NASH fatty livers. Characteristics NASH non fatty (n=16) NASH fatty (n=2) Age (years) 57.2 ± 2.6 57.9 ± 1.8 Gender, male: female 7/9 6/14 MELD score 26.8 ± 2.1 3.2 ± 1.8 Total bilirubin (mg/l) 9.8 ± 3.2 13.8 ± 3.8 Creatinine (mg/l) 3.9 ±.7 2.1 ±.31 Albumin (g/dl) 2.8 ±.14 3. ±.14 AST (U/L) 74.4 ± 14.9 66.4 ± 6.8 ALP (U/L) 225.5 ± 38.1 16.3 ± 2.9 Data are presented as means ± sem. MELD, model for end-stage liver disease; AST, aspartate transaminase, ALP; alkaline phosphatase. P<.5 versus NASH non-fatty by Students unpaired t-test. 4

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