room temperature for 40 min followed by addition of 20 ml of 1.5 N KOH (made in MeOH) and incubation at 37uC for 30 min. The pH was adjusted by adding 20 ml of 1.74 M acetic acid (made in MeOH) and 40 ml of the sample were assayed by HPLC [71]. Complex sphingolipids were analyzed as described previously [72] except for three changes. First, cells were grown in SDC medium (pH 6, 3.69 mM Fe) from 0.005 to 1 A600 nm units to produce uniform radiolabeling. Second, the concentration of [2-3H]myoinositol (American Radiochemicals, Inc., catalog #ART116) in the growth medium was 40 mCi/ml. Finally, each lane on the thin-layer plate contained 20 mL of non-radioactive yeast sphingolipids (0.5 mg/ml), which after charring, were used to verify the location of radioactive sphingolipids. Protein extraction and Western blotting analysis Cells were grown as described for a CLS assay and cell-free yeast extracts were prepared by using a modified published procedure [73]. Ten A600 nm units of cells were treated with cold TCA as described above, suspended in 400 ml of water followed by the addition of 400 ml of 0.2 M NaOH. Thoroughly mixed samples were incubated 5 min at room temperature, concentrated by centrifugation and suspended in 200 ml of lysis buffer (0.06 M Tris-HCl, pH 6.8, 5% glycerol, 2% SDS, 1% b-mercaptoethanol). After heating at 95uC for 5 min, samples were centrifuged and the protein concentration of the supernatant fluid was determined by using the DC protein assay kit (Bio-Rad Laboratories). Samples (50 mg of protein) electrophoresed on a 9% SDS-PAGE gel were transferred for 1.5 hours onto a PVDF membrane (Millipore Immobilon) by using a Bio-Rad semi-dry transfer system. For the blots shown in Figure 5I and 5J the acrylamide-bisacrylamide ratio was changed from the normal 37.5:1 ratio to 172:1. Membrane blocking and antibody binding were done in TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.5) containing 5% nonfat dry milk. Membranes were incubated for 2 hrs at room temperature with custom made polyclonal rabbit anti-Sch9 antibodies (Batch 2872, 1:1000, OpenBiosystems), phosphor- specific anti-Sch9T570 antibodies (1:10,000 dilution) [20], anti- HA antibodies (clone 3F10, 1:5000, Roche) or commercial monoclonal mouse anti-Vma2 (1:3,000 dilution) or anti-Vph1 (1:1000 dilution) antibodies (Molecular Probes). Other antibodies included alkaline phosphatase-linked anti-rabbit or anti-mouse IgG (1:5000, Sigma-Aldrich). Fluorescent signals from membranes exposed to an ECF substrate (Amersham Biosciences) were analyzed by using a PhosphorImager (Amersham Biosciences) and quantified by using ImageQuaNT Software. References 1. Powers RW, 3rd, Kaeberlein M, Caldwell SD, Kennedy BK, Fields S (2006) Extension of chronological life span in yeast by decreased TOR pathway signaling. Genes Dev 20: 174–184. 2. Harrison DE, Strong R, Sharp ZD, Nelson JF, Astle CM, et al. (2009) Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature 460: 392–395. 3. Bjedov I, Toivonen JM, Kerr F, Slack C, Jacobson J, et al. (2010) Mechanisms of life span extension by rapamycin in the fruit fly Drosophila melanogaster. Cell Metab 11: 35–46. 4. Blagosklonny MV (2009) Validation of anti-aging drugs by treating age-related diseases. Aging (Albany NY) 1: 281–288. 5. Kaeberlein M (2010) Resveratrol and rapamycin: are they anti-aging drugs? Bioessays 32: 96–99. 6. Goldberg AA, Richard VR, Kyryakov P, Bourque SD, Beach A, et al. (2010) Chemical genetic screen identifies lithocholic acid as an anti-aging compound that extends yeast chronological life span in a TOR-independent manner, by modulating housekeeping longevity assurance processes. Aging (Albany NY) 2: 393–414. 7. Fabrizio P, Pozza F, Pletcher SD, Gendron CM, Longo VD (2001) Regulation of longevity and stress resistance by Sch9 in yeast. Science 292: 288–290. Sphingolipids Control Lifespan Supporting Information Text S1 Supporting information, including supporting figures, table, and references. Figure S1: Outline of sphingolipid metabolism in Saccharomyces cerevisiae. Metabolic intermediates and complex sphingolipids are shown in bold font, genes are shown in italics and enzyme names are in regular lettering. Structures of compounds have been presented previously [74,75]. Figure S2: Myriocin treatment decreases cell size. DBY746 cells were grown with and without myriocin (Myr) as in a CLS assay using SDC medium (pH 4.5, 3X iron). After 72 hrs of incubation, cells were stained directly with Calcofluor white M2R (25 m/ml) and photographed at room temperature by using a Nikon Eclipse E600 fluorescence microscope equipped with a Plan Apo 1006 1.40 oil immersion objective, a SPOT RT 9.0 Monochrome-6 camera and SPOT basic software. For measurements, we excluded extremely large or small cells and cell diameter was calculated by measuring and averaging the long and short axes (perpendicular to each other) of each cell as described previously [30]. The median diameter for one hundred cells is indicated by a horizontal bar in the scatter plot. Figure S3: CLS of WT (DBY746) cells grown in SDC medium (pH 4.5, 3X iron) with CR (0.5% glucose) or without CR (NR, 2% glucose) +/2 myriocin (Myr) treatment. Data represent the mean 6 SEM of survival (* p,0.05, ** p,0.01, No Myr vs 450 or 600 ng/ml Myr, CR cultures). Figure S4: Sphingolipids activate the Pkh1/2 protein kinases. (A) Growth sensitivity was measured by diluting cells from CLS day 1 (10-fold serial dilution from left to right), spotting onto YPD plates containing the indicated concentration of myriocin, and incubating 3 days at 30 uC. Strains are: WT (R1158, LCB1), tetO7-LCB1 (RCD956), tetO7-LCB1/pkh1D (RCD1048), and tetO7-LCB1/pkh2D (RCD1051). (B) Same strains as used in A, but spotted onto YPD plates containing Dox. Table S1: Strains used in this study. (DOCX) Acknowledgments We thank Drs. R. Loewith, W. Longo, F. Madia, Y. Pan, G. Shadel, J. Urban, and M. Wei for yeast strains and advice on experimental procedures and Brad Withers for technical assistance. Author Contributions Conceived and designed the experiments: XH RCD. Performed the experiments: XH JL. Analyzed the data: XH JL RCD. Contributed reagents/materials/analysis tools: RCD XH JL. Wrote the paper: RCD. 8. Kaeberlein M, Powers RW, 3rd, Steffen KK, Westman EA, Hu D, et al. (2005) Regulation of Yeast Replicative Life Span by TOR and Sch9 in Response to Nutrients. Science 310: 1193–1196. 9. Selman C, Tullet JM, Wieser D, Irvine E, Lingard SJ, et al. (2009) Ribosomal protein S6 kinase 1 signaling regulates mammalian life span. Science 326: 140–144. 10. Kapahi P, Chen D, Rogers AN, Katewa SD, Li PW, et al. (2010) With TOR, less is more: a key role for the conserved nutrient-sensing TOR pathway in aging. Cell Metab 11: 453–465. 11. Hannun YA, Obeid LM (2008) Principles of bioactive lipid signalling: lessons from sphingolipids. Nat Rev Mol Cell Biol 9: 139–150. 12. Maceyka M, Milstien S, Spiegel S (2009) Sphingosine-1-phosphate: the Swiss army knife of sphingolipid signaling. J Lipid Res 50 Suppl: S272–276. 13. Stancevic B, Kolesnick R (2010) Ceramide-rich platforms in transmembrane signaling. FEBS Lett 584: 1728–1740. 14. Merrill AH, Jr., Wang MD, Park M, Sullards MC (2007) (Glyco)sphingolipidol- ogy: an amazing challenge and opportunity for systems biology. Trends Biochem Sci 32: 457–468. 15. Dickson RC (2008) Thematic review series: sphingolipids. New insights into sphingolipid metabolism and function in budding yeast. J Lipid Res 49: 909–921. PLoS Genetics | www.plosgenetics.org 14 February 2012 | Volume 8 | Issue 2 | e1002493