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HEP_25537_sm_SuppFig1.tif3942KSupplementary Figure 1. Effect of deletion of S6K1 or S6K2 on S6 phosphorylation, PPARa activity, and PPARa target gene expression in response to nutrients. (A) Subcellular localization of S6 phosphorylation in S6K1-/- or S6K2-/- mice. WT, S6K1-/-, or S6K2-/- mice were fasted for 24 h or fed ad libitum. Liver extracts were fractionated into cytosol and nucleus, and then analyzed by immunoblotting with the indicated antibodies. Blots are representative of two independent experiments. (B) Total serum ketones from WT or S6K1-/- mice that were fed or fasted for 24 h (n=7 per condition). (C) HepG2 cells were transfected with PPARa expression plasmids and non-silencing (NS) control or si-S6K1, along with PPRE reporter plasmid as indicated. Cell lysates were obtained 48 h after transfection, and luciferase activity was measured (n=4 per condition). Knockdown of S6K1 was determined by western blotting. Blots are representative of two independent experiments. (D) Quantitative real-time PCR from liver extracts of WT or S6K1-/- mice that were fed or fasted for 24 h. The data were normalized relative to L32 mRNA levels (n=4 per condition). Values are mean ± SEM. *P < 0.05; **P < 0.01; ns, not significant.
HEP_25537_sm_SuppFig2.tif1370KSupplementary Figure 2. Effect of S6 knockdown in ketone body production. HepG2 cells transfected with NS control or si-S6 were placed in vehicle or sodium octanoate for 3 days. Total ketones were determined from culture medium (n=3 per condition). Knockdown of S6 was determined by western blotting. Blots are representative of two independent experiments. ns, not significant.
HEP_25537_sm_SuppFig3.tif1371KSupplementary Figure 3. Effect of S6K2 knockdown on the transcriptional activity of PPAR families. (A), (B) HepG2 cells were transfected with PPARß/d (A) or PPARγ2 (B) expression plasmids and NS control or si-S6K2, along with PPRE reporter plasmid as indicated. Cell lysates were obtained 48 h after transfection, and luciferase activity was measured (n=3 per condition). Values are mean ± SEM. *P < 0.05; **P < 0.01; ns, not significant.
HEP_25537_sm_SuppFig4.tif1372KSupplementary Figure 4. Effect of S6K1 or S6K2 overexpression on ketone body production. (A), (B) HepG2 cells were transfected with empty vector (EV), PPARa, S6K1, or S6K2 expression plasmids as indicated, and were then treated with vehicle or sodium octanoate for 3 days. Total ketones were measured from culture medium (n=3). The overexpression of each gene was confirmed by western blotting (bottom). Blots are representative of two independent experiments. Values are mean ± SEM. *P < 0.05; **P < 0.01; ns, not significant.
HEP_25537_sm_SuppFig5.tif1370KSupplementary Figure 5. S6K2, but not S6K1, physically interacts with PPARa or NCoR1. Mouse liver extracts were immunoprecipitated using anti-S6K1 or S6K2 antibodies and assessed by immunoblotting with indicated antibodies. IgG served as a negative control. Blots are representative of two independent experiments.
HEP_25537_sm_SuppFig6.tif3940KSupplementary Figure 6. Subcellular localization of S6K2 and PPARa target gene expression in response to nutrient levels. (A) HepG2 cells were incubated in medium devoid of serum and glucose or in complete medium containing 10% FBS for 24 h and were then immunostained with anti-S6K2 antibodies and DAPI to label nuclei. Cell imaging was assessed by confocal microscopy. (B) HepG2 cells were incubated in medium devoid of serum and glucose or complete medium containing 10% FBS for 24 h; cell lysates were then fractionated into cytosol and nucleus, and analyzed by immunoblotting with the indicated antibodies. Blots are representative of two independent experiments. (C) Quantitative real-time PCR was performed on HepG2 cells that were incubated in medium devoid of serum and glucose or were incubated in complete medium containing 10% FBS for 24 h. The data were normalized relative to ß-actin mRNA levels (n=3 per condition). Values are mean ± SEM. *P < 0.05; **P < 0.01.
HEP_25537_sm_SuppFig7.tif1371KSupplementary Figure 7. Phosphorylation status of S6K2 in genetic model of obesity. (A) Validation of the antibody specificity against S6K1 or S6K2. Liver extracts form WT, S6K1-/-, or S6K2-/- mice were co-immunoprecipitated using anti-S6K1 or S6K2 antibodies and assessed by immunoblotting with anti-p70S6K T389 antibodies. (B) Liver extracts from WT or ob/ob mice were analyzed by western blotting with the indicated antibodies. Phosphorylation of S6K1 or S6K2 was detected using antibody against phospho-p70S6K T389 after immunoprecipitation with anti-S6K1 or S6K2 antibody. Each blot is representative of two independent experiments.
HEP_25537_sm_SuppTab1.doc45KSupplementary Table 1. Primers used for quantitative real time PCR.
HEP_25537_sm_SuppInfo.doc60KSupplementary Information.

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