Supplemental Figures Supplemental Figure 1. Fasting-dependent regulation of the SREBP ortholog SBP-1 and lipid homeostasis mediated by the SIRT1 ortholog SIR-2.1 in C. elegans. (A) Wild-type or sir-2.1(lof) fed or fasted animals stained with Sudan Black were photographed. Images were blinded as to genotype or nutritional status and scored for staining intensity. Graph represents data from three independent experiments. (B) Sudan Black staining of control (rol-6) or sir- 2.1OE were quantitated as in (A). 1
Supplemental Figure 2. Lipid storage in fasted sir-2.1(lof) animals requires sbp-1. (A) Animals were placed in fasting or fed conditions at 25 C then stained with Sudan Black. (B) Sudan Black staining was quantitated as in Supplemental Figure 1A. 2
Supplemental Figure 3. Lipid storage in fasted sir-2.1(lof) animals occurs independently of nhr-49. Animals were placed in fasting or fed conditions, then stained with Sudan Black. Sudan Black staining was quantitated as in Supplemental Figure 1A. 3
Walker et al._supplemental Figure 4 Supplemental Figure 4. SIR-2.1 is essential for proper fasting-dependent down-regulation of SBP-1::GFP C. elegans. (A) Fasting-dependent decreases in nuclear SBP-1::GFP in intestinal cells can be reversed by inhibition of sirtuins. Error bars represent standard deviation. White arrows represent positions of nuclei as visualized by light microscopy. Punctate cytoplasmic bodies visible in fasted intestines are autofluorescent gut granules that are unrelated to GFP expression. (B) dsir2 represses dsrebp target genes in wild-type Drosophila larvae. Prolonged fasting for 24 hours resulted in decreased expression of known dsrebp target genes such as acetyl-coa carboxylase (dacc), acetyl-coa synthase (dacs), and fatty acid synthase (dfasn) by qrt-pcr, while the dsir2-null larvae had higher expression of dsrebp target genes even in well-fed states (n=3). Single asterisks represent p-values less then 0.05, and double asterisks less than 0.01. 4
Supplemental Figure 5. Mammalian SIRT1 represses SREBP-1 and SREBP-2 target gene expression. (A) SREBP- 1 targets in the fatty acid biosynthesis pathway are upregulated in livers from SIRT1 liver-specific knockout mice. Target gene expression was analyzed by qpcr and normalized to -actin. Bars represent data from individual mice, ranked from low to high expression for each target gene. Me1: malic enzyme 1; ACLY: ATP-citrate lyase, ACSS1: acetyl-coa synthetase; ACACA: acetyl CoA-carboxylase. (B) SREBP-2 targets in the cholesterol pathway are upregulated in livers from SIRT1 liver-specific knockout mice. Expression of SREBP isoforms were not significantly changed. Target gene expression was analyzed by qrt-pcr and normalized to -actin. Bars represent data from individual mice, ranked from low to high expression for each target gene. MVD: melvalonate PP decarboxylase; SQLE: squalene epoxidase. 5
Supplemental Figure 6. Multiple mechanisms of sirtuin inhibition regulate nuclear abundance of SREBP-1a and SREBP-2 in HeLa cells. HeLa cells were placed in 1% lipid-depleted serum and proteasome inhibitors, then treated with nicotinamide or sirtinol for 6 hours. Cells were stained with antibodies to (A) SREBP-1a or (B) SREBP-2 along with DAPI and visualized by immunofluorescence. (C) HeLa cells were transfected with sirna to SIRT1 (Smartpool, Thermo-Fisher Scientific). At 42 hours, cells were treated with proteasome inhibitors, and were harvested at 48 hours, stained with antibodies to SREBP-1a along with DAPI and visualized by immunofluorescence. SIRT1 knockdown was assessed by qrt-pcr with SIRT1-specific primers. (D) Performed as (C) with antibodies to SREBP-2. (E) Overexpression of dominant negative SIRT1 (H363Y) increases SREBP-1a nuclear localization. HeLa cells were transfected for 42 hours, then treated with proteasome inhibitors for 6 hours. Cells were co-stained with antibodies for SREBP-1a, SIRT1 and DAPI. A transfected cell expressing high levels of SIRT1 H363Y are marked with a white arrow. DAPI positions for nontransfected cells are indicated by yellow arrows. 6
Supplemental Figure 7. Treatment of IMR-90 cells with SIRT1 activators or inhibitors did not significantly alter p300 HAT activity. p300 was immunopurified from IMR-90 cells treated with the SIRT1 activator SRT2183, the SIRT1 inhibitor nicotinamide (NAM) or vehicle control. Histone acetyltransferase (HAT) activity was determined by incubating high (++) or low (+) amounts of p300 with purified histones and radiolabeled acetyl-coa. The amount of p300 in each assay was determined by immunoblotting, as indicated. Acetylation of both histone H3 and H4 by p300 was detected by SDS-PAGE/fluorography, but was not affected by SRT2183 or NAM (compare lanes 1 and 2 (control) to lanes 3 and 4 (SRT2183) or lanes 5 and 6 (NAM). No HAT activity was identified in control IPs using an unrelated antibody (lanes 7 9). Ponceau S staining of total histones is shown and the position of each histone band indicated. 7
Supplemental Figure 9. Model for SIRT1 control of SREBP during feeding and fasting. 8