alkaline ceramidase, has been shown to increase lifespan in Drosophila [66]. As mutations and chromosomal rearrangements promote mammalian tumor cell formation, our demonstration that myriocin treatment reduces the rate of mutation and gross chromosomal rearrangements in yeast (Figure 5D and 5E) could lead to novel preventive measures and treatments for human cancers and thereby enhance lifespan. Materials and Methods Yeast strains, media, and plasmids Yeast strains used in our studies are listed in Table S1 in Text S1. Viability assays were done by using YPD agar plates (2% yeast extract, 1% peptone, 2% dextrose and 2% agar). For CLS and stress resistance assays, cells were grown in SDC medium supplemented with a 4-fold excess of the tryptophan, leucine, uracil and histidine to avoid possible artifacts due to auxotrophic deficiencies of the strain [30]. For initial experiments, SDC medium was lightly buffered to pH 6.0 (1.6 gm NaH2PO4 N H2O/liter), while in later experiments it was more strongly buffered with succinic acid (200 mM, final pH 4.5). Initially the concentration of iron in SDC medium was 1.23 mM (16 yeast nitrogen base) but in later experiments the concentration was raised 3-fold to avoid iron-starvation and reduced viability. In summary, SDC medium, pH 6.0, 16 iron, was used for the experiments shown in Figure 1, Figure 2, and Figure 3A–3F, while the same medium, but with 36 iron, was used for the experiments shown in Figure 3H, Figure 4, and Figure 6A. For the experiments shown in Figure 3G, 3H, Figure 5, Figure 6C– 6E, Figure 7, and Figures S2, S3, S4 in Text S1 the medium was SDC, pH 4.5, 36 iron. The pRS315 and pRS316 vectors [67] were used to make pRS315ADE2 by ligating a 2.3 Kb PstI-SpeI fragment carrying ADE2 into the corresponding sites of pRS315. Similarly, a genomic EcoRI-XhoI fragment was ligated to pRS316 cut with the same enzymes to make pRS316-PKH1 and a genomic BamHI- SalI fragment was ligated to pRS316 cut with the same enzymes to make pRS316-PKH2. pLCB1-5 was made by ligating a 4 Kb NruI-SacI chromosomal fragment containing LCB1 and its promoter [68] to pRS315ADE2 digested with SacI and NaeI (made blunt ended). pRS315-LCB2-B7 was made by ligating a 7 Kb BamHI chromosomal fragment containing LCB2 and its promoter [69] to pRS315 digested with BamHI. The plasmid encoding HA-tagged Dot6 has been described [36]. Lifespan assays CLS was measured as previously described [30]. Drug treatments were performed in medium containing a final concentration of 0.3% ethanol (ethanol plus drug). This was done be first adding a calculated volume of 95% ethanol to the medium followed by diluting a stock solution of myriocin (200 mg/ml in 95% ethanol, Sigma, # M1177) or doxycycline (100 mg/ml in 50% ethanol, Sigma, # D9891) to yield the desired concentration of drug (indicated in figure legends). Drugs were stored at 220uC and warmed to room temperature before dilution into medium, followed by thorough mixing. Cells grown overnight at 30uC in SDC medium were diluted into 25 ml of medium (125 ml flask) to give an initial A600 nm of 0.002 or as indicated in figure legends. Cultures were incubated at 30uC in an air bath shaker (220 rpm) for three days and cell viability was measured (CLS day 1) by diluting and spreading cells on YPD plates. After 2–3 days of incubation at 30uC colonies were counted and expressed a fraction of the day 1 value. Statistical Sphingolipids Control Lifespan significance was determined by using the two-tailed Student’s t-test. Stress resistance and oxygen consumption assays Cells grown in SDC medium for the time indicated in figure legends were diluted to an A600 nm of 1.0 in K-phosphate buffer (pH 6.0), treated with hydrogen peroxide for 60 min at room temperature with mixing, serially diluted (10-fold), spotted onto YPD plates, and incubated at 30uC for 2–3 days. For thermal stress resistance, cells were serially diluted (10-fold dilution starting with an A600 nm = 1), spotted onto YPD plates, and incubated at either 55uC (heat-shocked) or 30uC (control) for 45–180 min. Plates were incubated for 2–3 days at 30uC. For acetic acid resistance, cells from day 1 (72 hrs of incubation) of a CLS assay were treated with acetic acid as specified in the legend to Figure 6 for 2 hours at 30uC, followed by dilution and plating on YPD plates to measure colony forming units (CFUs). CFUs of the treated samples were expressed as a percentage of the untreated samples. Oxygen consumption was measured on cells grown from an A600 nm of 0.005 to 1 at which time the cell concentration was quantified by counting cells in a Petroff-Hausser counting chamber, cell size was measured as described in Supplemental Information and oxygen consumption was measured by using an Oxytherm System [38]. Data are expressed as nmol of oxygen consumed per min per total volume of 106 DBY746 cells. Age-dependent DNA damage measurements The frequency of spontaneous canavanine-resistant mutants (Canr) mutants was measured by using a published assay [42]. Cells were grown as described for CLS experiments and assayed for CFUs except that cultures were started at a density of 0.05 A600 nm units/ml and the SDC medium was buffered with succinate (200 mM, pH 4.5) and contained 3.69 mM iron (36 normal). These changes were necessary to prevent parental cells from dying so rapidly that they behaved like sgs1 mutant cells. Mutant sgs1 cells were grown in medium containing 300 ng/ml of myriocin. The frequency of Canr mutants was measured on the days indicated in Figure 6D. To identify Canr mutants, a sample of the culture was centrifuged and the cell pellet was washed once with sterile water and plated on selection medium (SDC-ARG supplemented with 60 mg/ml L-canavanine sulfate)s. After 3–4 days of incubation at 30uC, the colonies were counted and expressed as the ratio to total viable cells. GCRs were assayed as described previously [42]. Strains carried a URA3 cassette at the HXT13 locus which is located 7.5 kb from the CAN1 gene towards the telomeric of chromosome V. The experimental protocol was similarly to the one for measuring Canr mutations but the frequency of simultaneous loss of CAN1 and URA3 was measured by plating cells on SDC plates containing 1 mg/ml 5-fluoroorotic acid (5FOA) and 60 mg/ml L-canavanine sulfate [70]. Analysis of LCBs, LCBPs, and complex sphingolipids To extract LCBs and LCBPs, cells were grown as described for CLS assays and at the indicated time points 10 A600 nm units of cells were treated by slowly adding cold TCA to a final concentration of 6%. After incubating on ice for 5 min, cells were collected by centrifugation, washed twice with cold water, and suspended in 500 ml of fresh 80 mM triethylamine (made in HPLC grade absolute ethanol). Samples were sonicated in a water bath for 5 min at 37uC, heated for 30 min at 65uC, centrifuged and the supernatant fluid (150 ml) was transferred to an HPLC sample vial insert. AQC (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) reagent (30 ml) was added and samples were treated at PLoS Genetics | www.plosgenetics.org 13 February 2012 | Volume 8 | Issue 2 | e1002493