Physical and genetic interactions. The “Mitochondrial function” class had the highest number of genes in both the resistant and sensitive datasets, reflecting the broadly recognized importance of mitochondrial control for yeast apoptosis. We also found that metabolism is a major regulator of cell death, since impairment of major carbohydrate and amino acid metabolic pathways resulted in increased resistance to acetic acid-induced apoptosis. In addition, several other novel putative targets for the control of acetic acidinduced PCD were uncovered.MethodsStrainsThe parental strain Saccharomyces cerevisiae BY4741 (MATa his31 leu20 met150 ura30) and the respective EUROSCARF collection of derived deletion mutant strains, containing all the non-essential open reading frames replaced by the KanMX cassette, were used (http:// www-sequence.stanford.edu/group/yeast_deletion_project/ deletions3.html).Screening of the mutant collection for cell death susceptibility and resistanceCells were initially grown in 96-dot arrays on YPDA medium (yeast extract (1 ), bacto-peptone (2 ), glucose (2 ) and agar (2 )) for 48 h. Then, using a 96-pin replicator, strains were transferred into 96-well plates with YPD (yeast extract (1 ), bacto-peptone (2 ) and glucose (2 )), and grown for an additional 24 hours at 30 (no agitation) to be used as inocula. Each strain was then diluted 100 fold in YPD medium using a multichannel pipette (Additional file 1: Figure S1). Afterwards, again using a multichannel pipette, 2 l were transferred to new 96-well plates containing 150 L of YPD medium adjusted toSousa et al. BMC Genomics 2013, 14:838 http://www.biomedcentral.com/1471-2164/14/Page 3 ofpH 3.0 with HCl, and with acetic acid at a final concentration of 400 mM. A 2 M stock solution of acetic acid prepared with distilled water and adjusted to pH 3.0 with NaOH was used. At different times of incubation (100, 300 and 350 minutes), a 96-pin replicator was used to transfer a drop (approximately 3 l transfer volume) from each well into new 96-well plates containing YPD medium without acetic acid, and the plates were incubated at 30 for 48 hours. Optical density (OD640 nm) of the cultures was then read in a microplate reader (SpectraMax Plus, Molecular Devices) and the absence of any increase in OD640 nm was interpreted as indicative of the absence of viable/culturable cells.DSG3 Protein Molecular Weight Optical density of the 24 h-growth inocula was approximately the same for all the strains, ensuring that the same cell concentration was used in the treatment plates for all strains.PMID: 30176247 Optical density of the dilution plates was also read after 48 h of growth to control for any variation in final OD, and to confirm that all strains grew to the same extent without acetic acid treatment.PMID: 30176247 PI staining, chromatin condensation and fragmentation assessment, and detection of phosphatidylserine externalizationMicrosystems DM-5000B epifluorescence microscope coupled to a Leica DCF350FX digital camera, and at least 200 cells per experiment were counted.Cell viability ?CFU assaysCells were grown overnight in YPD medium in an orbital shaker, at 30 , 200 rpm to OD640nm 0.5. The strains were then harvested and suspended (107 cells/ml) in the treatment medium consisting of YPD at pH 3.0 (set with HCl) containing 100 mM acetic acid, and incubated in an orbital shaker, at 30 , 200 rpm. After 100 minutes of treatment, culture samples were taken, diluted, spread on YPDA plates and incubated at 30 . Cell viability was determine.