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Figure S1. Target analysis predicts that SIRT1 is a potential target of miR-146b.

MicroRNA target prediction databases, such as microRNA.org, showed alignment between miR-146b and SIRT1.

Figure S2. Knockdown of SIRT1 increased lipid accumulation in differentiated 3T3-L1 cells.

A. SIRT1 was knocked down by lentiviral shRNA-SIRT1 (Lenti-sh SIRT1) and confirmed by Western blot analysis. Lenti-sh NC, lentiviral shRNA negative control.

B. Preadipocytes transduced with Lenti-sh NC- or Lenti-sh were stimulated to differentiation. At day 8, differentiated cells were stained with Oil red O.

C. Intracellular lipid accumulation was quantified by measuring optical absorbance at 500 nm (n = 3). **p < 0.01. Values are means ± SD.

D. Preadipocytes that were transduced with Lenti-sh NC or Lenti-sh SIRT1 were transfected with miR-146b In or miR In CTL and stimulated to differentiate. Expression levels of miR-146b and SIRT1 were quantified by qRT-PCR on day 8 of differentiation (n = 3). Values are means ± SD. *p < 0.05 versus miR In CTL.

Figure S3. SIRT1 mediates the adipogenic effect of miR-146b in 3T3-L1 cells.

A. The effect of EX-527 on the anti-adipogenic activity of miR-146b In. Cells were pretreated with 10 μM EX-527 for 6 h. Media were then replaced with differentiation media containing 10 μM EX-527. Cells were stained with Oil red O on day 8 of differentiation, and lipid droplets were quantified (n = 3). Values are means ± SD. *p < 0.05 versus miR In CTL.

B. The effect of EX-527 on miR-146b expression in the experiment described in (A). Expression of miR-146b was measured by qRT-PCR (n = 3). Values are means ± SD. *p < 0.05 versus miR In CTL.

C. The effect of SA-3 on miR-146b expression. 3T3-L1 cells were transfected with miR-146b Ac or miR-Ac CTL. After 2 days, cells were maintained in growth media containing 5 μM SA-3 for 8 days. Expression levels of miR-146b were measured by qRT-PCR (n = 3). Values are means ± SD. *p < 0.05 versus miR Ac CTL.

D. The effect of SA-3 on SIRT1 mRNA expression in the experiment described in (C). Expression levels of SIRT1 mRNA were measured by qRT-PCR (n = 3). Values are means ± SD. *p < 0.05 versus miR Ac CTL.

Figure S4. Expression of miR-146b and its direct target, SIRT1 in WAT from various obese mice.

A–C. The increase in miR-146b and the resulting decrease in SIRT1 are correlated with hypertrophy of adipose tissue from obese mice, such as ob/ob (A), db/db (B) and diet-induced obese mice (C). qRT-PCR analysis was performed to measure miR-146b and SIRT1 mRNA expression levels in epididymal adipose tissue (n = 5). Values are means ± SEM. *p < 0.05 versus corresponding control mice.

Figure S5. Efficacy and toxicity of LNA-miR-146b antagomir.

A. Expression levels of miR-146b were measured by qRT-PCR in various tissues to test the efficacy of LNAmiR-146b antagomir in mice. Expression of miR-146b was compared between mice treated with LNA-miR-146b and those treated with LNA-scrambled negative control (n = 5). Values are means ± SEM. EF, epididymal fat pad; PF, perirenal fat; BF, brown fat; SF, subcutaneous fat; SI, small intestine; LI, large intestine.

B, C. Effect of LNA-miR146b injection on AST and ALT, which are biomarkers of liver injury (n = 5). Values are means ± SEM.

Figure S6. Knockdown of miR-146b reduced adiposity and whole body mass in high fat-fed obese mice.

A. Knockdown of miR-146b by LNA-miR-146b significantly decreased the acetylated FOXO1(Ac-FOXO1)/total FOXO1 ratio of white adipose tissue. Ac-FOXO1/FOXO1 ratio was based on densitometric quantifications of Ac-FOXO1 and total FOXO1 levels on Western blots (n = 5). Values are means ± SEM. *p < 0.05 versus LNA-scramble.

B. Knockdown of miR-146b by LNA-miR-146b reduced white adipose fat mass. The weight of perirenal white adipose tissue was measured 72 h after the last injection (n = 5). Values are means ± SEM. *p < 0.05 versus LNA-scramble.

C. Administration of LNA-miR-146b inhibitor (LNA-miR-146b) significantly decreased whole body mass (n = 5).

Values are means ± SEM. ns, not significant; *p < 0.05.

D. LNA-miR-146b significantly reduced total body fat mass compared to LNA-scramble (n = 5). Values are means ±SEM. ns, not significant; *p < 0.05.

Figure S7. Knockdown of miR-146b improved hepatic steatosis via upregulation of SIRT1 in high fat-fed obese mice.

A. Administration of LNA-miR-146b reduced hepatic hypertrophy (n = 5). Values are means ± SEM. *p < 0.05 versus LNA-scramble.

B. Injection of LNA-miR-146b decreased intracellular lipid accumulation in hepatocytes. Hepatic tissues were stained with H&E for histological examination. Scale bar = 100 μm.

C. Immunoblot showing SIRT1 and β-actin expressions.

D. Densitometric analyses for (C). Values are means ± SD. **p < 0.01.

Figure S8. Knockdown of miR-146b ameliorated insulin resistance in high fat-fed obese mice.

A. Glucose tolerance test in LNA-scramble and LNA-miR146b injected mice (n = 6). Values are means ± SEM. *p < 0.05 versus LNA-scramble.

B. Insulin tolerance test in LNA-scramble and LNA-miR146b injected mice (n = 6). Values are means ± SEM. *p < 0.05 versus LNA-scramble.

Table S1. Changes in the expression levels of various miRNAs after adipogenesis in 3T3-L1.

Table S2. Measurement of body weight and white adipose tissue (WAT) weight.

Table S3. Serum lipids profile in LNA-injected mice.

Table S4. Blood glucose and hormones levels in LNA-injected mice.

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