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hep26272-sup-0001-suppfig1.tif811KS1. Adenovirus-mediated overexpression of Thrsp in HepG2 cells. A) Quantitative PCR analysis showing a significant increase of Thrsp transcripts in cells infected with Ad-Thrsp at doses of 20 and 100 MOI. B). Western blot assay demonstrating a significant increase of Thrsp protein levels. MOI, multiplicity of infection.
hep26272-sup-0002-suppfig2.tif963KS2. Hepatic Thrsp overexpression increases liver lipid content and FAS, ACC and G6pase activity. A) Western blot assay demonstrates tail-vein injection of Ad-Thrsp resulted in a significant increase in hepatic Thrsp protein levels in C57Bl/6 mice. B) Experimental animal CT scan study showed liver lipid content was significantly increased after Thrsp overexpression. Enzymatic activities of hepatic FAS (C), ACC (D) and G6pase (E) were significantly increased in the livers with Thrsp overexpression. N=5,*P < 0.05 vs. Ad-GFP.
hep26272-sup-0003-suppfig3.tif747KS3. Plasma lipid levels in C57Bl/6 mice with hepatic Thrsp overexpression by adenovirus (Ad)-Thrsp. A) Plasma triglyceride (TG) levels 3 days after Ad-Thrsp administration. B) Plasma total cholesterol (CHO) levels 3 days after Ad-Thrsp administration. The results are presented as mean ± SEM. *p<0.05, n=5.
hep26272-sup-0004-suppfig4.tif876KS4. Thrsp overexpression decreases hepatic fatty acid uptake. A) 30 minutes post 18F-FTHA intravenous injection, the level of 18F-FTHA was determined by ?-counter and hepatic 18F-FTHA uptake in mice of Ad-Thrsp group was significantly lower than that of Ad-GFP group. N=4,*P < 0.05 vs. Ad-GFP. B) MicroPET imaging of each group showing decreased hepatic 18F-FTHA uptake in mice with liver Thrsp overexpression.
hep26272-sup-0005-suppfig5.tif727KS5. Thrsp overexpression promotes hepatic fatty acid β oxidation. Expression levels of genes involved in hepatic fatty acid β oxidation were examined via quantitative real-time PCR. Values are presented as mean ± SEM. *P<0.05 vs. Ad-GFP, n=4.
hep26272-sup-0006-suppfig6.tif1708KS6. Knockdown of hepatic Thrsp gene by siRNA ameliorates fatty liver in db/db mice. A) Hepatic Thrsp and FAS protein levels. *P < 0.05 versus db/m, #P < 0.05 versus scrambled siRNA, n=3. B) Hepatic triglyceride content. *P < 0.05 versus db/m, #P < 0.05 versus scrambled siRNA, n=3. C) Macroscopic appearance (left panels) and Oil Red O staining (right panels) of the liver of a representative db/m mouse (upper panels) and a db/db mouse receiving scrambled siRNA (middle panels) or Thrsp siRNA (lower panels) treatment.
hep26272-sup-0007-suppfig7.tif804KS7. Effect of hepatic Thrsp gene knockdown on markers of liver injury and inflammation in db/db mice. A) Serum ALT and AST levels. B) Quantitative PCR analysis of inflammatory gene expression. The results are expressed as the mean±SEM. *p<0.05 vs. db/db mice receiving scrambled siRNA treatment, n=3.
hep26272-sup-0008-suppfig8.tif1349KS8. Thrsp gene knockdown attenuates TO901317-induced lipid accumulation in cultured primary mouse hepatocytes. A) Immunoblot assay showing Thrsp gene silencing abolished TO901317-induced Thrsp expression. *P<0.05 vs. DMSO treatment; #p<0.05 vs. TO901317 treatment, n=5 B) Red oil O staining showing TO901317-induced hepatocyte lipid accumulation was significantly attenuated by Thrsp gene knockdown. *P<0.05 vs. DMSO treatment; #p<0.05 vs. TO901317 treatment, n=5 C) Quantitative PCR analysis of gene expression of SREBP1, FAS and ACC. TO901317 treatment significantly upregulated gene expression of SREBP1, FAS and ACC. Thrsp gene knockdown significantly attenuated TO901317-induced ACC expression and slightly reduced SREBP1 and FAS expression. *P<0.05 vs. DMSO treatment; #p<0.05 vs. TO901317 treatment, n=5 Scr: scrambled siRNA; si-Thrsp: Thrsp specific siRNAs.
hep26272-sup-0009-suppfig9.tif1356KS9. TO901317 treatment induces hepatic lipid accumulation. A). Changes of organ weight in control and TO901317-treated mice (5mg/kg/day for 3days). B). Liver sections were stained with Oil red O to reveal intracellular neutral lipids under light microscopy. C). Liver triglyceride content in control and TO901317-treated mice. D). Liver cholesterol content in control and TO901317 treated mice. Values are presented as mean ± SEM. ***P<0.001, vs. Control; n=5.
hep26272-sup-0010-supptab1.doc59KSupplemental Table 1. Formula and fatty acid composition of control (10% fat) and high fat (45%fat) diets.

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