Yeast strains, plasmids, and growth conditions

All experiments were done with isogenic Saccharomyces cerevisiae strains in the W303-1B background (MATα ade2, his3, leu2, trp1, ura3, ssd1-d2), and with Saccharomyces boulardii (Florastor, Lot No. 538) obtained from Biocodex, Inc. (San Bruno, CA). For all the experiments described in this paper, cells were cultured and treated using standard yeast protocols [41]. Unless noted otherwise, all other drugs and reagents were purchased from SIGMA-Aldrich.

Ethanol-induced cell death assay

Cells of the indicated strain and genotype were cultured in rich YPD media overnight, resuspended in fresh media, and allowed to reach exponential phase (an approximate OD 600 value of 0.2). They were then resuspended in water or fresh media or in water or fresh media containing either 15% or 22% ethanol [33], and allowed to grow at 30°C for the indicated times. Next, they were either serially diluted onto YPD plates and cultured at 30°C for 2 days to test for viability or treated with the appropriate stain for the indicated test, and examined using a Zeiss LSM 700 Confocal Laser Scanning Microscope. At least three independent cultures were tested and compared. Statistical significance was determined with the Student’s t-test.

Acetic acid-induced cell death assay

Cells of the indicated genotype were cultured in rich YPD media overnight, resuspended in fresh media, and allowed to reach exponential phase (an approximate OD 600 value of 0.2). They were then resuspended in fresh media pH 3 or fresh media pH 3 containing 160mM acetic acid, allowed to grow at 30°C with shaking for 2 hours. Next, they were treated with the appropriate stain for the indicated test, and examined using a Zeiss LSM 700 Confocal Laser Scanning Microscope.

Hydrochloric acid-induced cell death assay

Cells of the indicated genotype were cultured in rich YPD media overnight, resuspended in fresh media, and allowed to reach exponential phase (an approximate OD 600 value of 0.2). They were then resuspended in water, water containing either 50 mM or 75 mM HCl, water containing 50 mM HCl and 2 mM AdoMet, or water containing 2 mM AdoMet alone. They were allowed to sit at room temperature for 1.5 hours. Then, they were either serially diluted onto YPD plates and cultured at 30°C for 2 days to test for viability or treated with the appropriate stain for the indicated test, and examined using a Zeiss LSM 700 Confocal Laser Scanning Microscope.

Viability assay

Cells were grown overnight in rich YPD media at 30°C and then diluted to a final concentration (an approximate OD 600 value of 0.2). For each strain, a series of 10-fold dilutions was then prepared in water over a range of concentrations from 10-1 to 10-5 relative to the initial culture. Spots of 5 μl from each dilution series were then plated on the indicated media and cultured at 30°C for 2 days. Individual colonies were then counted and compared to the number of colonies observed from an untreated culture serially diluted at the beginning of the experiment. Several serial dilutions for each culture were done to ensure that there were enough colonies to count for statistical significance and at least three independent cultures were tested and compared. Statistical significance was determined with the Student’s t-test. Note that after 3 hr, cells cultured in rich media without any cell death inducing agents were able to grow and to divide, hence the relative viability levels that are greater than 100%.

In vivo detection of mitochondrial fragmentation, ROS accumulation, and caspase activation

Mitochondrial fragmentation was detected in S. boulardii cells using 10 nM Mitotracker Green (Molecular Probes), according to the manufacturer’s specifications. Intracellular ROS accumulation was examined after treatment with 5 μg/ml of dihydrorhodamine 123 (DHR123; Sigma Aldrich) [42]. Activated caspase-like activity was detected in S. boulardii cells after treatment using a FLICA apoptosis detection kit (ImmunoChemistry Technologies, LLC) according to the manufacturer’s specifications [43, 44]. After exposure to reagents, S. boulardii cells were harvested and examined using a Zeiss LSM 700 Confocal Laser Scanning Microscope.

Fluorescence microscopy

Cells were grown to mid-log phase in selective media and examined using a 63X oil-immersion objective and a pinhole size of 1 Airy Unit using a Zeiss LSM 700 Laser Confocal Microscope Images were captured and processed using the ZEN 2009 software package.

Microarray experiments: array design

Genomic sequences were obtained from the Saccharomyces Genome Database (downloaded from http://www.yeastgenome.org). These sequences were used to design a custom 8×15K array using the Agilent eArray software (http://earray.chem.agilent.com/). Each array had a minimum of 2 unique 60-mer probes designed against 6,612 open reading frames encoded by S. cerevisiae. This resulted in a total of 13,275 unique probes for each array, including Agilent hybridization controls.

Microarray experiments: sample preparation, extraction, and purification

S. boulardii cells were cultured in rich YPD media overnight, resuspended in fresh media, and allowed to reach exponential phase (an approximate OD 600 value of 0.2). They were then resuspended in 45 mL of either water, for the control condition, or water containing 50 mM HCl for the experimental condition. The total number of cells in each experiment was 3 × 108, as measured with a spectrophotometer. After a 1.5 hr incubation with shaking at room temperature, the cells were washed with 1x PBS, frozen in liquid nitrogen, and stored at −80°C. Total RNA was then extracted using a RiboPure Yeast Kit (Ambion) and purified of gDNA with Turbo DNase (Ambion). RNA was assessed using a NanoDrop-2000c spectrophotometer (Thermo Scientific) and Agilent 2100 bioanalyzer to determine RNA concentration, purity, and integrity.

Microarray experiments: cDNA synthesis, labeling, and hybridization

cDNA was generated from 10 μg aliquots of purified RNA by first annealing hand-mixed random oligonucleotides (pdN9, 6.3 μg) and oligo(dT) 19 V (8.3 μg) obtained from IDT (Integrated DNA Technologies). First strand cDNA synthesis was then performing using Super Script III reverse transcriptase (Invitrogen) in a reaction containing 0.25 mM DTT and 0.5 mM total deoxynucleoside triphosphates (amino-allyl-dUTP and deoxynucleoside triphosphates) in a ratio of 3:2 aa-dUTP. After synthesis for 3 hr at 42°C, the cDNA was hydrolyzed with 0.3 M NaOH and 0.03 M EDTA. The reaction was then neutralized with 0.3 M HCl to pH 7.0. Following this, cDNA was purified using a 25 ug capacity DNA Concentrator and Cleanup Kit (Zymo), dried using a Speed-vac, resuspended in ddH 2 O (2 μg cDNA per 9 μl water), and stored at −80°C. Dye coupling was achieved by adding 1 μL of 1.0 M NaHCO 3 solution (pH 9.0) and 1.25 μL of either Cy3 or Cy5 Amersham monoreative dye (GE Healthcare; dissolved in DMSO) to each 9 μL aliquot of cDNA, then incubating for 1 hr at room temperature in darkness. Unincorporated dye was removed and the samples purified using the Zymo cleanup kit. Dye incorporation and cDNA yield were quantified using the NanoDrop-2000c spectrophotometer on the microarray setting. 300 ng of the relevant Cy3- and Cy5-stained cDNAs (control and experiment) were then pooled in a total volume of 25 μL ddH 2 O and denatured at 95°C for 3 min. Following denaturation, 25 μL of 2x HiRPM gene expression and hybridization buffer (Agilent) was added to each sample. These cDNA solutions were then applied to the microarray slide and incubated at 65°C for ~17 hr in a hybridization oven, as per the manufacturer’s instructions. The slides were then sequentially washed in a row of Agilent Wash Buffer I, Agilent Wash Buffer II, and acetonitrile (Sigma), and dried using Agilent drying and stabilization buffer.

Microarray data analysis and bioinformatics

Slides were scanned using an Axon 4000B scanner (Molecular Devices) and fluorescence was quantified using GENE Pix Pro 3.0 software (Molecular Devices). Data was then normalized using the Goulphar transcriptome platform (http://transcriptome.ens.fr/goulphar/). Duplicate spots for each gene were averaged in Microsoft Excel, and the results were confirmed using qPCR. The Cytoscape 2.8.3 (http://www.cytoscape.org/%20download.php) plugin BiNGO 2.44 was used to identify enriched biological processes in differentially expressed genes after Benjamini & Hochberg false discovery correction for multiple hypothesis testing. Pairwise average linkage clustering analysis was performed using the program Cluster and visualized using Treeview [45].