length of the G1 phase accurately, by calculating two parameters: i) the ‘‘critical size’’ these newborn daughter cells must attain to initiate cell division; ii) the rate (‘‘growth rate’’) at which they grow to their critical size. DMSO-treated cells had a critical size of 63.262.4 fl and a specific growth rate constant k = 0.32860.008 h21 (Figure 4). Rapamycin markedly prolonged the G1 phase, because cells had to reach a substantially larger critical size (79.461.2 fl) before they could initiate DNA replication (Figure 4A). Rapamycin-treated cells also grew very slowly (k = 0.10460.004 h21, Figure 4B), although this effect was evident ,1 h after addition of the drug (Figure S1). We found that cells treated with gemfibrozil delayed initiation of DNA replica- tion, not because they had altered critical size (65.460.6 fl, Figure 4A), but because they reached that size slower than cells treated with DMSO did (k = 0.28760.07 h21, P = 0.005, Figure 4B). In addition, from the cell size distributions of asynchronously dividing cells, we obtained the ‘‘birth size’’ of newborn cells (see Materials and Methods). While DMSO-treated cells had a ‘‘birth size’’ of 40.362.7 fl under these growth conditions, gemfibrozil-treated newborn cells were significantly smaller (30.164.7 fl, P = 0.04, Figure 4C). Taken together, these data show that the smaller ‘‘birth size’’ and slower ‘‘growth rate’’ of cells treated with gemfibrozil lengthen the G1 phase. Next, we focused on the effects of gemfibrozil and fluoxetine on overall cell proliferation rates. We tested these drugs alone and in combination, at several doses (Figure 5A). We found that gemfibrozil did not significantly affect overall cell proliferation at the doses tested (Figure 5). Hence, the prolongation of the G1 phase by gemfibrozil is likely accompanied by compensatory shortening of subsequent cell cycle phases, resulting in similar overall generation time. On the other hand, fluoxetine arrested proliferation of yeast cells at 200 mM (Figure 5A, first green bar to the left; and Table S3, bottom left cell). To our knowledge, the near complete inhibition of yeast cell proliferation by fluoxetine has not been reported. Remarkably, however, addition of gemfibrozil even at a 4-fold less molar concentration fully suppressed the inhibitory effects of fluoxetine (see Figure 5A, compare the left green bar to the other green bars; and Table S3, last row). We then added the two drugs not simultaneously, but in different order, removing the first drug before adding the second (Figure 5B). We found that gemfibrozil suppressed fluoxetine’s anti-proliferative effects only if added before (representative experiment in Figure 5B, compare the blue and yellow bars on the right; and Table S4, compare the top and middle cells in the 3rd column), but not after fluoxetine (Figure 5B, compare the left and middle green bars; and Table S4, compare the left and middle cells in the 3rd row). These results suggest that the suppressive interaction between gemfibrozil and fluoxetine is not due to extracellular interaction or competition for transport between the two drugs. Furthermore, the results from the order-of addition experiment suggest that gemfibrozil acts upstream, since it does not reverse fluoxetine’s inhibition of cell proliferation. Instead, it appears that fluoxetine cannot inhibit cell proliferation in the context of gemfibrozil’s prior action. Understanding the basis of the interaction between gemfibrozil and fluoxetine requires a mechanistic understanding of their function in yeast cells. We examined the combined effects on cell proliferation between gemfibrozil and fluoxetine because of the novel cell cycle effects of each compound, affecting different phases of the cell cycle. We would like to note, however, that the suppressive interaction between the two compounds could be unrelated to their cell cycle effects. For example, gemfibrozil might induce expression of proteins that do not interfere with cell cycle DNA Content Analysis and Drug Interactions progression, but may cause fluoxetine resistance. Fluoxetine is an anti-depressant thought to act as a serotonin-specific reuptake inhibitor [18]. Hence, the effects we described for fluoxetine in yeast appear to result from some other mechanism. Similarly, nuclear receptors of the PPARa/RXR type, the target of gemfibrozil, are thought to be unique to animals and sponges [19,20], but ancestral analogs may exist in yeast [21]. Nonetheless, although the effects of fluoxetine and gemfibrozil on yeast cells we described above likely represent off-target modes of action, they may act similarly in other eukaryotic organisms, including humans. In conclusion, our results suggest that monitoring the effects of drugs on cell cycle progression reveals unexpected cellular roles of widely prescribed compounds. Finally, although we did not test all possible combinations of the compounds that affected cell cycle progression, at least in the case of gemfibrozil and fluoxetine, our results suggest that combining such com- pounds may also be an effective strategy to identify novel drug interactions. Materials and Methods Yeast strains For our primary analysis, we used the S. cerevisiae strain JTY2953 (MATa pdr5::TRP1 snq2::hisG ade2-101 his3-D200 leu2-D1 lys2- 801am trp1-D63 ura3–52; a generous gift from Dr. Paul deFigueir- edo, Texas A&M University). For the elutriation experiments in Figure 4 we used the diploid strain BY4743 (MATa/a his3D1/ his3D1 leu2D0/leu2D0 lys2D0/LYS2 MET15/met15D0 ura3D0/ ura3D0; commercially available from Open Biosystems). For all other experiments, we used the haploid strain BY4741 (MATa his3D1 leu2D0 met15D0 ura3D0; commercially available from Open Biosystems). Media and culture conditions In all experiments, strains were cultured at 30uC in YPD (1% yeast extract, 2% peptone, 2% dextrose). For our primary analysis with the JTY2953 strain, overnight cultures were diluted 1:200 and aliquoted into 96-well plates, 198 ml per well. To each well we then added 2 ml of a drug stock solution (2 mg/ml in DMSO), resulting in a final drug concentration of 20 mg/ml. At the four corner wells of each 96-well plate, the cultures were treated with DMSO only. These cultures served as the mock-treated reference samples. The plates were then placed at 30uC and incubated for 6–7 h. Each of the 200 ml cultures were then transferred to microcentrifuge tubes containing 500 ml ethanol, and sonicated for 5 s. For the experiments where the drugs were added in dividing JTY2953 cells, the overnight cultures were diluted 1:400 and incubated for 3 h at 30uC. We then added the drugs of interest and incubated the plates at 30uC for another 6 h before fixing the samples in ethanol. For DNA content measurements in BY4741 cells, which proliferate faster than JTY2953 cells do, overnight cultures were diluted 1:400, cultured for 2.16 h before we added the drugs of interest, and then cultured for another 4.33 h before they were fixed in ethanol. Cell size determinations To obtain size distributions from asynchronous cultures, overnight cultures of BY4743 cells were diluted 1:500 in fresh medium, and incubated for 2 h at 30uC. We then added the drugs of interest and incubated at 30uC for another 4 h. Cell size was then measured with a Beckman Z2 Channelyzer. For each sample we analyzed, we obtained size distributions from two different dilutions of cells. The average of the geometric mean of each size PLoS ONE | www.plosone.org 5 May 2012 | Volume 7 | Issue 5 | e36503