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Additional Supporting Information may be found in the online version of this article.

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hep26368-sup-0001-suppinfo.doc59KSupplementary Information
hep26368-sup-0002-supptables.doc163K

Supporting table 1. Serum biochemical and cytokine, hormone analysis and liver function analysis kits.

Supporting table 2. Primers for Real-time PCR detection.

Supporting table 3. Antibodies for immunoblot analyses.

Supporting table 4. The primers for making constructs.

hep26368-sup-0003-suppfig1.tif151KSupporting Figure 1. IRF9 deficiency does not affect food intake but enhances hepatic gluconeogenesis. (A) Energy of food intake by wild-type and IRF9 KO mice fed with the normal chow or HFD was calculated every week. n=28-39 per group. (B) For all the statistical significance is indicated and compared with the WT HFD group, **p < 0.01.
hep26368-sup-0004-suppfig2.tif278KSupporting Figure 2. IRF9 deficiency aggravates the hepatic steatosis upon HFD feeding. (A) Liver function was examined by measuring the levels of alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) in the serum by reagent kits, n=7 per group. (B) Immunoblot analysis indicated the inhibition of AMPK signaling in liver extracts from IRF9 KO mice. Protein expression levels were quantified and normalized to loading control GAPDH. All values are expressed as the mean ± SEM. The statistical significance is indicated and compared with the WT HFD group,**p<0.01, ***p < 0.001; compared with the WT NC group, ##p < 0.01.
hep26368-sup-0005-suppfig3.tif157KSupporting Figure 3. IRF9 deficiency does not affect lipid metabolism in WAT. Relative mRNA levels of representative adipogenesis associated genes (PPARγ, C/EBPα, C/EBPδ, pref-1 and Cyclin D1), and lipogenic genes (GPAT, FAS and SREBP-1c) and genes responsible for fatty acid oxidation (PPARα, ACOX and CPT-1α) and lipolyisis (HSL and ATGL) in epididymal fat extracts from IRF9 KO and WT mice. n=6-12 per group. All values are expressed as the mean ± SEM.
hep26368-sup-0006-suppfig4.tif466KSupporting Figure 4. Hepatic IRF9 overexpression improves metabolism in diet-induced obese mice. (A) The levels of TG, cholesterol and NEFA were extracted in liver tissue from HFD group between WT with KO mice. n= 5 each group. (B) Liver function was examined by measuring the levels of ALT, AST and ALP in the serum by reagent kits, n=6 per group. (C) The mRNA levels of proinflammatory and anti-inflammatory markers in liver were measured by real-time PCR. n=12 for each group. All values are expressed as mean ± SEM. The statistical significance is indicated and compared with the GFP adenovirus injected group, *p < 0.05, **p < 0.01.
hep26368-sup-0007-suppfig5.tif476KSupporting Figure 5. Hepatic IRF9 overexpression improves metabolism in ob/ob mice. (A) The levels of TG, cholesterol and NEFA were extracted in liver tissue from HFD group between WT with KO mice. n= 5 each group. (B) Liver function was examined by measuring the levels of ALT, AST and ALP in the serum by reagent kits, n=7 per group. (C) The mRNA levels of proinflammatory and anti-inflammatory markers in liver were measured by real-time PCR. n=12 for each group. All values are expressed as mean ± SEM. The statistical significance is indicated and compared with the GFP adenovirus injected group, *p < 0.05, **p < 0.01.
hep26368-sup-0008-suppfig6.tif433KSupporting Figure 6. IRF9 interacts with PPARα to activate PPARα target genes. (A) The mRNA levels of PPARα target genes in the primary mouse hepatocytes transfected with GFP or IRF9 plasmid are examined by real-time PCR. (B) The mRNA levels of PPARα target genes in the primary mouse hepatocytes transfected with GFP or mutant IRF9 plasmid of which the PPARα-interaction domain was deleted. (C) The mRNA levels of PPARα target genes in the liver of WT mice fed with HFD injected with adenovirus containing GFP or IRF9. (D) The mRNA levels of PPARα target genes in the liver of ob/ob mice injected with adenovirus containing GFP or IRF9. In (A) to (D), values are presented as the mean ± SEM,and statistical significance is indicated and compared with the Ad-GFP group, *p < 0.05, **p < 0.01.
hep26368-sup-0009-suppfig7.tif901KSupporting Figure 7. Hepatic PPARα overexpression rescues deregulated metabolism in IRF9 KO mice. (A) The mRNA levels of PPARα target genes in primary mouse hepatocytes infected with adenovirus expressing IRF9 or vector controls. (B) Immunoblot showed the PPARα expression in livers four weeks after jugular vein injection of adenovirus within PPARα cDNA or GFP sequence control. (C) The mRNA levels of PPARα and its target genes were determined by real-time PCR. (D) The levels of TG, cholesterol and NEFA were extracted in liver tissue from HFD group between WT with KO mice. n= 5 for each group. (E) Liver function was examined by measuring the levels of ALT, AST and ALP in the serum by reagent kits, n=7-9 per group. (F and G) The mRNA levels of proinflammatory and anti-inflammatory markers in livers of WT and IRF9 KO mice were measured by real-time PCR. n=9-12 for each group. All values are expressed as mean ± SEM. The statistical significance is indicated and compared with the GFP adenovirus injected WT group, *p < 0.05; compared with PPARα adenovirus injected WT group, +p<0.05; compared with the GFP adenovirus injected IRF9 KO group, #p< 0.05.

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