raw flow cytometry data in a public database (see Dataset S1, and Materials and Methods). To identify drugs that altered the cell cycle, we compared the frequency distribution of cultures treated with drugs against a normal distribution fit of the reference (n = 82) samples (Figure 3A). Samples that had a %G1 greater or less than two standard deviations from the mean of the reference sample distribution were considered to differ significantly from the mock-treated samples (Figures 1 and 3A). Drugs that led to an increase (%G1.60.00%) in the percentage of cells with unreplicated DNA formed the ‘‘High G1’’ group, while others led to a mitotic delay and a ‘‘Low G1’’ (%G1,38.76) DNA content (see Figure 3A, and Dataset S1). In this initial screen, we added the drugs to cultures diluted from an overnight stationary phase culture, where most cells would be in the G1 phase of the cell cycle [1]. Hence, drugs in samples with a ‘‘High G1’’ DNA content may have arrested cell cycle progression non-specifically. In that case, the high G1 DNA content reflected the state of the starting culture, and not cell cycle effects of the drugs. To exclude such possibilities, we re-tested the ‘‘High G1’’ drugs by adding them to actively dividing cells (see Figure 1). Overall, from this primary analysis we identified 27 compounds that interfered with progression in the G1 phase of the cell cycle, before initiation of DNA replication, resulting in a ‘‘High G1’’ DNA content (see Table S1). Another 12 drugs affected mitotic progression, resulting in a ‘‘Low G1’’ DNA content (see Table S2). Along with DNA content, we also analyzed the forward scatter (FSC) from the same flow cytometry experiments (see Figure 3B). FSC values often serve as a proxy for cell size, but they are also affected by cell shape and intracellular composition [8]. We noticed that most drugs in the ‘‘Low G1’’ group had elevated FSC values compared to the group with no cell cycle effects (Figure 3B). This is consistent with the notion that mitotic delay leads to an increase of cell size. It should also be noted that yeast cells in mitotic phases of the cell cycle are budded [1]. Hence, their irregular shape may also contribute to an increase in FSC values. An increase of FSC values was also evident for a significant fraction, but not all, of drugs in the ‘‘High G1’’ group (Figure 3B). We are not aware of other systematic studies of drug effects on cell cycle progression measured by DNA content analyses. Our results reveal that several drugs currently and commonly used for human therapy have specific effects on the eukaryotic cell cycle. The higher number of drugs that interfered with G1 progression likely reflects the fact that cells commit to initiation of cell division in the G1 phase [1,9,10]. Among the ‘‘High G1’’ group, we noted antifungals that inhibit biosynthesis of ergosterol, a component of fungal membranes [11], and rapamycin, a potent inhibitor of the TOR pathway known to block G1 progression [12]. Overall, however, there was a diverse range of compounds in the ‘‘High G1’’ group (see Table S1). Although most drugs in the ‘‘Low G1’’ group have well established mitotic roles (see Table S2), we noted that the highest-ranked drug from this group was fluoxetine (brand name Prozac). To our knowledge, this is the first time that such strong cell cycle effects have been reported for fluoxetine. Since we did our primary analysis in a sensitized pdr5D, snq2D yeast strain, we then tested the drugs that led to the ‘‘High G1’’ and ‘‘Low G1’’ groups against the PDR5+, SNQ2+ wild type reference strain BY4741. We found that several drugs were not effective in this case. For example, lovastatin, which leads to a G1 arrest in mammalian cells [13], had no effect in PDR5+, SNQ2+ yeast cells (see Table S1). This is consistent with an earlier report that yeast cells are sensitive to lovastatin in a pdr5D -dependent manner [14]. Nonetheless, about half of the drugs in both groups DNA Content Analysis and Drug Interactions Figure 3. DNA content analysis identifies drug effects on cell cycle progression. A, Cumulative histogram displaying the percent- age of cells in the G1 phase of the cell cycle (%G1), for cells treated with a panel of FDA-approved drugs. The bin width of the histogram is 1%, with each bin containing all the drugs with values within the bin boundaries. The black line superimposed to this histogram is the normal distribution fit of the %G1 values of the reference sample. Bins with values .2 sd from the mean of the wild type distribution are in grey (‘‘Low G1’’ group) and black (‘‘High G1’’ group). B, From all the samples we analyzed by flow cytometry, the %G1 is on the x-axis, and the forward angle scattering (FSC) values on the y-axis. We colored the data points of the sub-groups as in A. doi:10.1371/journal.pone.0036503.g003 remained effective in cells with intact multidrug transporters (see Tables S1 and S2). Among drugs that led to a ‘‘High G1’’ DNA content, we further examined the cell cycle effects of the potent antilipemic gemfibrozil [15], a Peroxisome Proliferator-Activated Receptor a (PPARa) agonist. To our knowledge, a G1 cell cycle role for gemfibrozil has not been reported, in any system. The High G1 DNA content could result from roles specific to G1 progression, or manifest in G1 as a ‘‘carryover’’ from roles in other cell cycle phases. To distinguish between these two possibilities, we added gemfibrozil to highly synchronous newborn G1 cells, obtained by centrifugal elutriation [16,17]. As a function of time, we then measured cell size and the percentage of budded cells (budding correlates with initiation of DNA replication in yeast [1]). This allowed us to measure the PLoS ONE | www.plosone.org 4 May 2012 | Volume 7 | Issue 5 | e36503