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Figure S1. LC3 expression was suppressed, but the activity of CTSB and CTSL were not impaired in the fatty liver. (A–B) (A) The final body weight and (B) liver weight after HFD feeding during the indicated periods. (C) Hepatic total lipid content was measured in livers of HFD-fed mice using the Folch method. (D) The liver was enlarged by long-term HFD feeding. Hematoxylin and eosin staining of the liver showed that lipid accumulation was induced by HFD feeding. Scale bar: 100 μm. (E) The levels of GOT1/AST (glutamic-oxaloacetic transaminase, soluble) and GPT/ALT (glutamic-pyruvic transaminase, soluble) were increased in the livers of long-term-HFD-fed mice (20 wk). (F) Representative images of Masson’s trichrome-stained liver of long-term HFD-fed mice (20 wk). Scale bar: 100 μm. (G) The mRNA levels of pro-inflammatory and anti-inflammatory cytokines and (H) lipid metabolism-related genes in the livers of the 20-week group. (I) LC3 protein levels in livers of HFD-fed mice in the presence or absence of protease inhibitor, leupeptin (Leup). Relative fold-change of LC3-II protein levels compared to the LFD group. The relative protein levels were quantified using ImageJ analysis software. (J) Protein levels of CTSB and CTSL in HFD-fed mouse liver. (K) Relative fold-change of CTSB and CTSL protein levels compared to the LFD group. (L) Activity of CTSL and CTSB in liver tissue. (M) Representative immunohistochemistry analysis of LC3 images. Scale bar: 100 μm. (N) Immunofluorescence analysis of LC3 images in the livers of HFD-fed mice. Scale bar: 10 μm. LC3 puncta were quantified using ImageJ threshold analysis and were expressed as a ratio relative to the number of LC3 puncta obtained for the LFD group (n = 10). Values represent mean ± SEM (n = 10). * p < 0.05; ** p < 0.01; *** p < 0.001, compared to the LFD group. Cd36: cluster of differentiation 36; Fasn: fatty acid synthase; HFD: high-fat diet; Il4: interleukin 4; Il6: interleukin 6; LFD: low-fat diet; Lipe/Hsl: hormone-sensitive lipase; Pnpla2/Atgl: patatin-like phospholipase domain containing 2; Ppara: peroxisome proliferator activated receptor alpha; Pparg: peroxisome proliferator activated receptor gamma; Scd1: stearoyl-Coenzyme A desaturase 1; SEM: standard error of the mean; Srebf1: sterol regulatory element binding transcription factor 1; Tgfb1: transforming growth factor beta 1; Tnf: Tumor necrosis factor. Figure S2. Mir214-3p bound to 3′ UTR sites of Atg7, Atg16l1, and Ulk2. (A) Heatmap of miRNA expression in the livers of HFD-fed mice. The miRNA levels were analyzed using a TLDA kit. The RNA samples from the livers were pooled by group, and PCR arrays were analyzed in triplicate. (B) Heatmap of autophagy gene expression in miRNA inhibitor-transfected cells. (C–D) Hepatocytes were transfected with (C) Mir214-3p or Mir145a-5p inhibitors and (D) mimics for 48 h; target autophagy gene mRNA levels were measured using qRT-PCR. (E) The prediction of the Mir214-3p binding site in the 3′ UTR of Atg7, Atg16l1, and Ulk2. (F) Hepa 1-6 cells were co-transfected with Mir214-3p mimic and the GLuc-SEAP vector containing the 3′ UTR of the indicated genes. After 48 h, the relative luciferase activity was measured in cell culture media, and the activity levels of Gaussia luciferase were normalized to those of secreted alkaline phosphatase. Values represent mean ± SD (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001, compared to the negative control (NC). HFD: high-fat diet; PCR: polymerase chain reaction; qRT-PCR: quantitative reverse transcription PCR; SD: standard deviation; TLDA: TaqMan Low Density Array.Figure S3. Expression of transcription factor genes in the fatty liver. (A) Difference between the means of expression levels at 5 and 10 weeks and those at week 20 are shown in the clustering map. (B) Hepa 1-6 cells were transfected with the indicated siRNA for 48 h, and the silencing effect was validated using qRT-PCR. (C) The levels of transcription factors known to regulate the expression of Ulk1 were measured using qRT-PCR. Values represent mean ± SEM (n = 6). * p < 0.05; *** p < 0.001, compared to the LFD group. (D–E) Hepa1-6 cells were transfected with (D) Atf3 siRNA or infected with (E) Ad-Atf3 virus particles for 48 h. The target autophagy gene mRNA level was measured using qRT-PCR. (F) Normal and Hnf4a-overexpressing hepatocytes were transfected with the Mir214-3p mimic. After 48 h, the protein levels of ATG7 were measured. Values represent mean ± SD (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001, compared to the negative control (siControl or Control). Ad-Atf3 virus: adenovirus expressing Atf3; Creb: cAMP responsive element binding protein; Foxo1: forkhead box O1; Foxo3: forkhead box O3; Nr1h4/Fxr: nuclear receptor subfamily 1, group H, member 4; HFD: high-fat diet; LFD: low-fat diet; Trp53/p53: transformation-related protein 53; PCR: polymerase chain reaction; qRT-PCR: quantitative reverse transcription PCR; SD: standard deviation; siRNA: small interfering RNA; Tfe3: transcription factor E3; Tfeb: transcription factor EB.Figure S4. Expression of Tfeb and Tfe3 was not regulated by Mir214-3p. (A-B) The levels of Tfeb, Tfe3, and Ulk1 in (A) Mir214-3p inhibitor-transfected or (B) mimic-transfected hepatocytes. Hepatocytes were transfected with Mir214-3p inhibitor or mimic for 48 h. Values represent mean ± SD (n = 3). ** p < 0.01; *** p < 0.001, compared to the negative control (NC). PCR: polymerase chain reaction; qRT-PCR: quantitative reverse transcription PCR; SD: standard deviation; Tfe3: transcription factor E3; Tfeb: transcription factor EB.Figure S5. Suppression of Mir214-3p levels increased autophagic activity by restoring Ulk1 levels, but did not affect the expression of lipid metabolism-related genes in the livers of HFD-fed mice. (A–B) (A) The organ weight and (B) Mir214-3p levels of epididymal white adipose tissue, kidney, and gastrocnemius muscle in anti-Mir214-3p-treated mice. Mir214-3p levels were systemically reduced, but there was no effect on organ weight. Values represent mean ± SEM (n = 5). ** p < 0.01; *** p < 0.001, compared to the LFD group. (C) Immunofluorescence analysis of LC3 in anti-Mir214-3p-treated liver tissue. Scale bar: 15 μm. Values represent mean ± SEM (n = 10). * p < 0.05; *** p < 0.001, compared to the LFD group. (D–E) (D) The mRNA levels and (E) protein levels of lipid metabolism-related genes in the liver. (F) Immunofluorescence analysis of ULK1 in anti-Mir214-3p-treated liver tissue. Scale bar: 30 μm. (G–H) The activity and expression of AMPK in (G) Mir214-3p inhibitor- or (H) mimic-transfected hepatocytes. AICAR (0.5 mM) and Compound C (Com C, 20 μM) were treated at 24 h after transfection, and protein levels were then analyzed after 24 h. (I) The Mir214-3p levels in AMPK activator (AICAR)-treated or inhibitor (Com C)-treated hepatocytes. (J) The levels of Mir214-3p and Ulk1 mRNA in Prkaa2-silenced hepatocytes. Values represent mean ± SD. Fasn: fatty acid synthase; HFD: high-fat diet; LFD: low-fat diet; PCR: polymerase chain reaction; Ppara: peroxisome proliferator activated receptor alpha; Pparg: peroxisome proliferator activated receptor gamma; Prkaa2/Ampk: AMP-activated protein kinase, alpha 2 catalytic subunit; qRT-PCR: quantitative reverse transcription PCR; Scd1: stearoyl-CoA desaturase-1; SEM: standard error of the mean; siRNA: small interfering RNA; Srebf1: sterol regulatory element binding transcription factor 1; TUBA: tubulin alpha; VCL: vinculin. Figure S6. Inhibition of Mir214-3p and Ulk1 expression reduced oleic acid-induced lipid accumulation. (A-B) Oil Red O staining of the negative control (anti-NC) or anti-Mir214-3p-transfected hepatocytes. At 24 h after transfection, cells were treated with oleic acid for 24 h to induce lipid accumulation in hepatocytes. The representative images of (A) Oil Red O-stained hepatocytes and quantitative analysis of the (B) relative intensity of the Oil Red O staining. (C) Immunofluorescence analysis of lipid droplets (BODIPY) and LC3 in Ulk1+/+ or ulk1-/- MEFs. Values represent mean ± SD (n = 5). * p < 0.05; ** p < 0.01; *** p < 0.001. BODIPY: boron-dipyrromethene; OA: oleic acid; SD: standard deviation.Table S1. Composition of the diet fed to the mice.IngredientLFD(g/kg)HFD(g/kg)Casein200200Corn oil5050Lard-200Cholesterol-5Corn starch350145Sucrose300300Cellulose5050Mineral3535Vitamin1010Methionine33Choline bitartrate22Total1,0001,000LFD: low-fat diet; HFD: high-fat dietTable S2. Total triglyceride (TG), total cholesterol (TC), GOT1 (glutamic-oxaloacetic transaminase 1, soluble) and GPT (glutamic pyruvic transaminase, soluble) levels in the human non-fatty and fatty liver tissues used in this study.TG (28–200)TC (120–220)GOT1 (10–40)GPT (5–40)Non-fatty liver tissuesN-122892026N-2942162213N-3124746939N-41622555981N-5451332641Fatty liver tissuesF-140224064250F-22981723990F-314015467142F-4159218170231F-5299264330434F-6212195321306These data were provided from Gachon University Gil Medical Center (Incheon, Republic of Korea) (GCIRB2016-277)Table S3. Primer sequences used in this study.GeneForward primer (5′→3′)Reverse primer (5′→3′)Pik3c3AACAACCGTGTCGCTCTTTGGAACCATCTGCCTCCACGTTAUlk1ACATCCGAGTCAAGATTGCTGGCTGGGACATAATGACCTCAGGAtg16l1AGCAGCTACGAGACGCTCTCGCATCGAAGACATACGAGGBecn1/Beclin1ATGGAGGGGTCTAAGGCGTCTCCTCTCCTGAGTTAGCCTCTAtg5TGTGCTTCGAGATGTGTGGTTGTCAAATAGCTGACTCTTGGCAAAtg7GTTCGCCCCCTTTAATAGTGCTGAACTCCAACGTCAAGCGGUlk2TCAATGCCTAGTTGGAAAACCACAGTAAGGTGATGTTTCTCTGGGAGabarapAAGAGGAGCATCCGTTCGAGAGGGGCTTTTTCCACTATCACCUvragACATCGCTGCTCGGAACATTCTCCACGTCGGATTCAAGGAAAmbra1GTCCACGCTCGACCTTCTTACCACTTGCCAGTCTTAACCTCTAtg10GTAGTTACCAAGTGCCGGTTCAGCTAACGGTCTCCCATCTAAAAtg3ACACGGTGAAGGGAAAGGCTGGTGGACTAAGTGATCTCCAGAtg12GGCCTCGGAACAGTTGTTTACAGCACCGAAATGTCTCTGASqstm1/p62ATGTGGAACATGGAGGGAAGAGGAGTTCACCTGTAGATGGGTHnf4a/Hnf4αGGTTTAGCCGACAATGTGTGGTCCCGCTCATTTTGGACAGCSmad9CGGGTCAGCCTAGCAAGTGGAGCCGAACGGGAACTCACTfebGCAGCCACCTGAACGTGTATGTTAGCTCTCGCTTCTGAGTTfe3TGCGTCAGCAGCTTATGAGGAGACACGCCAATCACAGAGATFoxo3CTGGGGGAACCTGTCCTATGTTCATTCTGAACGCGCATGAAGNr1h4/FxrGCTTGATGTGCTACAAAAGCTGCGTGGTGATGGTTGAATGTCCTrp53/p53GTCACAGCACATGACGGAGGTCTTCCAGATGCTCGGGATACCrebCTGAGAGCTGGTATGTCAGGATGAGTGCTGGAGTAAAACAGTCAActb/β-actinGCAGGAGTACGATGAGTCCGACGCAGCTCAGTAACAGTCCTable S4. Taqman probes used in this study.Assay nameAssay IDAssay nameAssay IDmmu-Mir-423-5p002340PIK3C3Hs00176908_m1mmu-Mir-193b-5p002467ULK1Hs00177504_m1mmu-Mir-27a-3p000408ATG16L1Hs01003142_m1mmu-Mir-125a-5p002198BECN1Hs01007018_m1mmu-Mir-324-5p000539ATG5Hs00169468_m1mmu-Mir-296-5p000527ULK1Hs00979043_m1mmu-Mir-27b-3p000409GABARAPHs00925899_g1mmu-Mir-29c-3p000587UVRAGHs01075434_m1mmu-Mir-34b-3p002618AMBRA1Hs00387943_m1mmu-Mir-99b-5p000436ATG10Hs00919718_m1mmu-Mir-100-5p000437ATG3Hs00223937_m1mmu-Mir-221-3p000524ATG12Hs01047860_g1mmu-Mir-132-3p000457SQSTM1Hs01061917_g1mmu-Mir-150-5p000473ATG7Hs00893766_m1mmu-Mir-145-5p002278SMAD9Hs00195441_m1mmu-Mir-214-3p002306HNF4AHs00230853_m1ATF3Hs00231069_m1ACTBHs01060665_g1Table S5. miRNA inhibitors, mimics, and siRNAs used in this study.Product nameCatalog numbermiRIDIAN microRNA mmu-miR-423-5p hairpin inhibitorIH-310823-02-0002miRIDIAN microRNA mmu-miR-193b-3p hairpin inhibitorIH-310816-02-0002miRIDIAN microRNA mmu-miR-27a-3p hairpin inhibitorIH-310523-07-0002miRIDIAN microRNA mmu-miR-125a-5p hairpin inhibitorIH-310392-08-0002miRIDIAN microRNA mmu-miR-324-5p hairpin inhibitorIH-310537-08-0002miRIDIAN microRNA mmu-miR-296-5p hairpin inhibitorIH-310476-07-0002miRIDIAN microRNA mmu-miR-27b-3p hairpin inhibitorIH-310380-07-0002miRIDIAN microRNA mmu-miR-29c-3p hairpin inhibitorIH-310522-08-0002miRIDIAN microRNA mmu-miR-34b-3p hairpin inhibitorIH-310929-02-0002miRIDIAN microRNA mmu-miR-99b-5p hairpin inhibitorIH-310387-07-0002miRIDIAN microRNA mmu-miR-100-5p hairpin inhibitorIH-310567-07-0002miRIDIAN microRNA mmu-miR-221-3p hairpin inhibitorIH-310583-08-0002miRIDIAN microRNA mmu-miR-150-5p hairpin inhibitorIH-310425-07-0002miRIDIAN microRNA mmu-miR-132-3p hairpin inhibitorIH-310406-07-0002miRIDIAN microRNA mmu-miR-145a-5p hairpin inhibitorIH-310422-08-0002miRIDIAN microRNA mmu-miR-214-3p hairpin inhibitorIH-310573-08-0002miRIDIAN microRNA mmu-miR-324-5p mimicC-310537-07-0002miRIDIAN microRNA mmu-miR-221-3p mimicC-310583-07-0002miRIDIAN microRNA mmu-miR-145a-5p mimicC-310422-07-0002miRIDIAN microRNA mmu-miR-214-3p mimicC-310573-07-0002ON-Targetplus mouse Hnf4a siRNA-SMARTpoolL-065463-00-0005ON-Targetplus mouse Smad9 siRNA-SMARTpoolL-046344-01-0005ON-Targetplus mouse Tfeb siRNA-SMARTpoolL-050607-02-0005ON-Targetplus mouse Tfe3 siRNA-SMARTpoolL-054750-00-0005ON-Targetplus mouse Atf3 siRNA-SMARTpoolL-058604-01-0005ON-TARGETplus Non-targeting siRNAD-001810-01-05 ................
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