Figure 1. Effects of FGF21 on adipose tissue. (A) Representative histological. findings of epididymal adipose tissue (B) mrna expression of

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1 SUPPLEMENTAL MATERIAL EN Figure 1. Effects of FGF21 on adipose tissue. (A) Representative histological findings of epididymal adipose tissue (B) mrna expression of adipocytokines in adipose tissue. A, db/m control; B, db/db + vehicle; C, db/db + FGF21. A H&E stain of adipose tissue. Original magnification X400. PPARγ, peroxisome proliferator-activated receptor γ; Adipo, adiponectin; MCP-1, monocyte chemoattractant peptide-1; PAI-1, plasminogen activator inhibitor-1; TNFα, tumor necrosis factor α; Data are expressed as mean ± SEM. *p<0.05, **p<0.01 vs. db/m control; ##p<0.01 vs. db/db + vehicle. Figure 2. Representative renal histological findings in experimental animals. A, F, K, PAS stain; B, G, L, type IV collagen stain; C, H, M, TGFβ-1 stain; D, I, N, fibronectin stain; E, J, O, laminin stain; A-E, db/m control; F-J, db/db + vehicle; K-O, db/db + FGF21. Original magnification X400. PAS, periodic acid-schiff. Figure 3. Basal FGF21 expression in various renal cells. (A) mrna expression of FGF21 in renal cells under basal conditions (B) Representative western blot for FGF21 in renal cells under basal conditions. MMC, mouse 1

2 mesangial cell; HK2, human proximal tubule cells; Podocytes, mouse podocytes. Data are means ± SEM. **p<0.01 vs. MMC. Figure 4. Effects of high glucose (30mM) stimuli on FGF21 expression in cultured mesangial cells. (A) Effects of HG on FGF21 mrna expressions. (B) Representative western blot for FGF21. NG, normal glucose; HG, high glucose; Data are expressed as mean ± SEM. Figure 5. Basal FGF21 signaling as determined by the activation level of ERK1/2 from renal cortical tissues in experimental animals. Representative western blot for phophospecific-erk1/2 and total ERK1/2. Figure 6. Profibrotic molecule synthesis in response to high-glucose (30mM) stimulation with or without stealth small interfering (si) RNA of FGF21 in cultured mesangial cells. (A) mrna expression of profibrotic molecules under high-glucose (30mM) stimulation with or without gene silencing for FGF21 (B) Representative western blot for FGF21, TGFβ1, type IV collagen, PAI-1 with or without transfection with sirna against FGF21 under high-glucose stimulation. MMCs were treated with 30mM D-glucose medium for 72 h with or without transfection with sirna for FGF21. NG, normal glucose (5mM); HG, high-glucose (30mM); sicontrol; Stealth RNAi 2

3 negative control duplexes; Data are means ± SEM. *p<0.05 vs. NG; #p<0.05 vs. HG. Figure 7. Cholesterol, triglyceride content and lipid peroxidation in renal cortical tissue in control db/m mice based on age. (A) Cholesterol content in renal cortical tissue (B) Triglyceride content in renal cortical tissue (C) Lipid hydroperoxides (LPO) content in renal cortical tissue Data are means ± SEM. *p<0.05, **p<0.01 vs. db/m control at 8weeks of age. 3

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11 Supplementary Table 1. Primer sequences for real-time quantitative PCR Target gene Primer sequence (5 to 3 ) Size (bp) MCP-1, forward CTGGATCGGAACCAAATGAG 95 MCP-1, reverse CGGGTCAACTTCACATTCAA PAI-1, forward TCCTCATCCTGCCTAAGTTCTC 365 PAI-1, reverse GTGCCGCTCTCGTTTACCTC TGFβ1, forward AGCCCGAAGCGGACTACTAT 96 TGFβ1, reverse CTGTGTGAGATGTCTTTGGTTTTC Col-IV, forward GCTCTGGCTGTGGAAAATGT 102 Col-IV, reverse CTTGCATCCCGGGAAATC Adiponectin, forward TGTTGGAATGACAGGAGCTGAA 121 Adiponectin, reverse CACACTGAAGCCTGAGCGATAC PPARγ, forward AGACCACTCGCATTCCTTTGACAT 271 PPARγ, reverse TCCCCACAGACTCGGCACTCAATG SREBP1, forward AAGATGTACCCGTCCGTGTC 80 SREBP1, reverse GGCTGTAGGATGGTGAGTGG HMG-CoA, forward AGCCGAAGCAGCACATGAT 201 HMG-CoA, reverse CTTGTGGAATGCCTTGTGATTG ABCA1, forward CGTTTCCGGGAAGTGTCCTA 246 ABCA1, reverse GCTAGAGATGACAAGGAGGATGGA FAS, forward CCTGGATAGCATTCCGAACCT 183 FAS, reverse Visfatin, forward Visfatin, reverse TNFα, forward TNFα, reverse FGF21, forward FGF21, reverse β-klotho, forward β-klotho, reverse FGFR1c, forward FGFR1c, reverse FGFR2c, forward FGFR2c, reverse FGFR3c, forward AGCCATCTCGAAGGCTACACA TGCCGTGAAAAGAAGACAGA ACTTCTTTGGCCTCCTGGAT CCGATGGGTTGTACCTTGTC GGCAGAGAGGAGGTTGACTTT GTCAAAGCCTCTAGGTTTCTTTGC GGCCTCAGGATCAAAGTGAG CAGGCCCATTGTTACCTTGT CTCCAAAGGTCTGGAAGCAG AATACCACCGACAAGGAAATGG AGTTACCCGCCAAGCACGTA GTTCTCTATATTCGGAATGTAACTTTTGAG AGG CGC TGG CAG AAC TGT ACG CCC TAC GTC ACT GTA CTC A FGFR3c, reverse GGT GAC ATT GTG CAA GGA CAG A FGFR4, forward GCTCGGAGGTAGAGGTCTTG 95 FGFR4, reverse GTAGGAAAGGCCGATGGAGT β-actin, forward β-actin, reverse GGACTCCTATGTGGGTGACG CTTCTCCATGTCGTCCCAGT 118 MCP-1, monocyte chemoattractant peptide-1; PAI-1, plasminogen activator inhibitor-1; TGFβ, transforming growth factor; Col-IV, type IV collagen; PPARγ, peroxisome proliferator-activated receptor γ; SREBP1, sterol regulatory element-binding protein-1; HMG-CoA, cholesterol 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase; ABCA1, ATPbinding cassette transporter-1; FAS, fatty acid synthase; TNFα, tumor necrosis factor α; FGF21, fibroblast growth factor 21; FGFR, FGF receptor; In this experiment, each sample was run in triplicate, and the corresponding non-reverse transcribed mrna samples were used as negative controls. The mrna level of each sample was normalized to that of β-actin mrna.

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