,300 and contained all events of sufficient contiguity as defined by the default autogating parameters, on average ,95% of total. From the gated populations, we determined the mean and standard deviation of the FSC parameter. Cell cycle phase subpopulations were computed from the gated population using the Dean-Jett-Fox model without constraints. %G1 was defined as the area of the G1 model peak, divided by the combined areas of the G1 and G2/M peaks. Because the %G1 and associated parameters represent continua across experiments, it was neces- sary to identify model fits that did not accurately represent experimental data. This was assessed primarily by root mean square (RMS) error and ratio of mean fluorescence intensities (MFI, calculated from the FL2-A parameter) of the G2/M vs. G1 peaks. Automated unconstrained analyses that yielded extremes in these parameters, or extremes in %G1 or S-phase components of the model fit, were manually constrained by application of the median G2/G1 MFI ratio and a G1 MFI position that minimized the resulting overall RMS. All model fits were visually inspected in order to confirm the accuracy of the fit. Unquantifiable data was excluded from further analysis. Experimental data and correla- tions are provided in the searchable spreadsheet available as Dataset S1. Raw data files can be freely accessed at Cytobank (www.cytobank.org) and are found in the public experiments ‘‘Yeast DNA Content Project - DRUG - INCLUDED’’ and ‘‘Yeast DNA Content Project - DRUG -EXCLUDED’’. Proliferation assays Yeast strain BY4741 was grown overnight at 30uC in a 1 ml YPD starter culture, then diluted 1:200 into fresh YPD in the presence or absence of drug. 200 ml volumes were aliquoted into clear flat-bottom 96-well sterile cell culture plates (Thermo Scientific, Nunc MicroWell Plate 167008), and the absorbance at 600 nm was measured hourly using a Tecan infinite 200Pro plate reader, after one minute of 3.5 mm orbital shaking to re- suspend cells. Plates were incubated standing at 30uC in between measurements. Absorbances were blanked post-measurement against wells containing media and DMSO alone. For combina- tion assays, cells were treated with drug at the time of initial reseeding, at a final DMSO concentration of 1.24% throughout, aliquoted immediately into 96-well plates for reading of absor- bance, and followed as described above. Growth constants were calculated using a best fit for exponential growth incorporating time points from 2 h through 6 h. For order of addition experiments, cells were reseeded at 1:200 into fresh YPD in a culture tube, cultured standing at 30uC with hourly re-suspension for 3 h, then divided into three tubes and treated with the first drug (200 mM) or DMSO-only control, at a final DMSO concentration of 0.62% throughout. Following an additional 3 h of incubation at 30uC, the primary treated cultures were washed twice with fresh YPD at 30uC, re-suspended in the same, and References 1. Pringle JR, Hartwell LH (1981) The Saccharomyces cerevisiae cell cycle. In: Strathern JD, Jones EW, Broach JR, eds. The molecular biology of the yeast Saccharomyces. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. pp 97–142. 2. Sharma SV, Haber DA, Settleman J (2010) Cell line-based platforms to evaluate the therapeutic efficacy of candidate anticancer agents. Nat Rev Cancer 10: 241–253. 3. Caminero JA, Sotgiu G, Zumla A, Migliori GB (2010) Best drug treatment for multidrug-resistant and extensively drug-resistant tuberculosis. Lancet Infectious Dis 10: 621–629. 4. Pirrone V, Thakkar N, Jacobson JM, Wigdahl B, Krebs FC (2011) Combinatorial approaches to the prevention and treatment of HIV-1 infection. Antimicrob Agents Ch 55: 1831–1842. 5. Huggins DJ, Venkitaraman AR, Spring DR (2011) Rational methods for the selection of diverse screening compounds. ACS Chem Biol 6: 208–217. DNA Content Analysis and Drug Interactions further divided for treatment with the second drug, as above, resulting in nine total temporal combinations of vehicle, gemfi- brozil, and fluoxetine. Growth constants were calculated as above from 0 h through 6 h. Drugs The FDA-approved library was purchased from Enzo (Cat. #: BML-2841). Artemisinin was from Enzo (Cat. #: ALX-350-219), gemfibrozil from Sigma (Cat. #: G9518), while chlorpromazine (Cat. #: 101077-482), fluoxetine (Cat. #: 89160-860) and clinafloxacin (Cat. #: 89150-368) were purchased through VWR International. All drug stock solutions were in DMSO. Supporting Information Figure S1 Determining the length of G1. Left, Graphs from which we determined the specific rate of cell size increase constant k, shown in Figure 4, from the same elutriation experiments. The natural log cell size (y-axis) is plotted against time (shown in hours, x-axis). Right, Graphs of the fraction of budded cells (y-axis) as a function of cell size (in fl, x-axis), from the same elutriation experiments. The data points shown were used to estimate the critical size for division we show in Figure 4A. In A, the cells were treated with DMSO, in B with rapamycin (at 0.1 mg/ml), and in C with gemfibrozil (at 50 mg/ml). (TIF) Drugs that lead to a High G1 DNA content. Drugs that lead to a Low G1 DNA content. Table S1 (DOCX) Table S2 (DOCX) Table S3 Fluoxetine strongly inhibits yeast cell prolif- eration, but it is suppressed by gemfibrozil. (DOCX) Table S4 Gemfibrozil suppresses fluoxetine’s anti-pro- liferative effects only if added before, but not after, fluoxetine. (DOCX) Dataset S1 Searchable spreadsheet of all the primary data, arranged in different worksheets. (XLSX) Author Contributions Conceived and designed the experiments: MP. Performed the experiments: SAH CD IM SE SCS SSD EMH KED RS MP. Analyzed the data: SAH CD IM SE SCS RS MP. Contributed reagents/materials/analysis tools: SAH RS MP. Wrote the paper: SAH RS MP. 6. Borisy AA, Elliott PJ, Hurst NW, Lee MS, Lehar J, et al. (2003) Systematic discovery of multicomponent therapeutics. Proc Natl Acad Sci USA 100: 7977–7982. 7. Haase SB, Lew DJ (1997) Flow cytometric analysis of DNA content in budding yeast. Methods Enzymol 283: 322–332. 8. Keller HU, Fedier A, Rohner R (1995) Relationship between light scattering in flow cytometry and changes in shape, volume, and actin polymerization in human polymorphonuclear leukocytes. J Leukocyte Biol 58: 519–525. 9. Johnston GC, Pringle JR, Hartwell LH (1977) Coordination of growth with cell division in the yeast Saccharomyces cerevisiae. Exp Cell Res 105: 79–98. 10. Pardee AB (1989) G1 events and regulation of cell proliferation. Science 246: 603–608. 11. Parks LW, Casey WM (1995) Physiological implications of sterol biosynthesis in yeast. Annu Review Microbiol 49: 95–116. PLoS ONE | www.plosone.org 8 May 2012 | Volume 7 | Issue 5 | e36503