Gemfibrozil delays initiation of DNA replication.

<p>A, The critical cell size (shown in fl) of diploid BY4743 cells treated with DMSO, rapamycin (0.1 µg/ml) or gemfibrozil (50 µg/ml), was measured from synchronous elutriated cultures, in YPD medium. The data points shown were from three independent experiments in each case. The <i>P</i> values shown were calculated from paired, two-tailed <i>t</i> tests, assuming unequal variance. The data used to calculate these parameters are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036503#pone.0036503.s001" target="_blank">Figure S1</a>. B, The specific rate of cell size increase constant <i>k</i> (in h⁻¹) was measured from the same elutriation experiments shown in a, assuming exponential growth. The data used to calculate these parameters are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036503#pone.0036503.s001" target="_blank">Figure S1</a>. C, The cell size distributions of the indicated cell populations, proliferating asynchronously in YPD medium, were measured using a channelyzer (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036503#s4" target="_blank">Materials and Methods</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036503#pone.0036503-Hoose1" target="_blank">[22]</a>). Cell numbers are plotted on the y-axis and cell size (in fl) on the x-axis. Daughter “birth” size was defined as the maximum size of the smallest 10% of cells on the left side of the cell size distribution of each sample <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036503#pone.0036503-Hoose1" target="_blank">[22]</a>.</p

Representative DNA content histograms.

<p>Independent experiments of the indicated samples are shown in each case. Fluorescence is plotted on the x-axis, while the number of cells analyzed is on the y-axis. Reference samples were treated with DMSO, shown at the top. Examples of “High G1” profiles include cells treated with ketoconazole or gemfibrozil, while cells treated with fluoxetine give rise to a “Low G1” DNA content profile. At the bottom, we show a few examples of complex DNA content histograms that were unquantifiable. These include profiles of cells treated with suramin and 5-fluorouracil (antineoplastic agents), and flubendazole (a microtubule blocker used as anti-nematodal).</p

Decision flow-chart diagram of our primary analysis.

<p>This diagram summarizes our DNA content measurements using the <i>pdr5Δ, snq2Δ</i> strain. See text for details.</p

DNA content analysis identifies drug effects on cell cycle progression.

<p>A, Cumulative histogram displaying the percentage 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.</p

Cys4p advances START both by promoting cell growth and by a separate function, which does not require CBS's catalytic activity.

<p>A, Rate of cell size increase (shown as growth rate, in fl/min) for the indicated strains was measured assuming linear growth from synchronous elutriated cultures in media that contain galactose and induce expression of the <i>P_GAL</i> alleles (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#s4" target="_blank">Methods</a>). The average value for each strain is shown with a horizontal bar (± sd). Where indicated, the <i>P</i> values shown were calculated from two-tailed <i>t</i> tests. The data used to calculate the values shown in A and B are in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590.s009" target="_blank">Figure S8</a>. B, The critical cell size of the indicated strains (shown in fl), was measured from the same elutriation experiments shown in A (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590.s010" target="_blank">Figure S9</a>). The analogous experiments in non-inducing, glucose containing, medium are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590.s010" target="_blank">Figure S9</a>.</p

Network representation of the “Low G1” group.

<p>The interactions shown are from the gold-standard reference database BioGRID <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590-Stark1" target="_blank">[54]</a>. The network was constructed with Cytoscape <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590-Smoot1" target="_blank">[83]</a>, and displayed using an unbiased, force-generated layout. Only the factors that showed interactions (physical or functional) are included. We also included the essential gene <i>CDC28</i> (shown in red), encoding the major yeast Cdk.</p

Decreased fitness correlates with altered cell cycle progression.

<p>The y-axis shows the fitness values of Giaever et al <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590-Giaever1" target="_blank">[33]</a>. Higher values indicate reduced fitness. The cutoff for reduced fitness was about <85% of the wild type in that study <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590-Giaever1" target="_blank">[33]</a>. Thus, strains with possible small reductions in fitness have been assigned a “WT-like” fitness score of 1. Giaever et al <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590-Giaever1" target="_blank">[33]</a> evaluated fitness of the same strains we used, during growth in rich (YPD-2%Dextrose) liquid media, allowing for a direct comparison with our dataset. We used the non-parametric Spearman test to obtain the correlation (<i>r</i>) values we show. The correlation coefficient for all the strains (<i>r</i>_T) is shown at the bottom right of the graph. We colored the r values for the sub-groups as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen-1002590-g002" target="_blank">Figure 2</a>. For every gene we included in this analysis, the values we used in this correlation are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590.s001" target="_blank">Dataset S1</a>.</p

Schematic overview of our approach.

<p>For a detailed description of all the protocols we used, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#s4" target="_blank">Methods</a>.</p

Interactions among the factors of the “High G1” group.

<p>The network of interactions was constructed and displayed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen-1002590-g007" target="_blank">Figure 7</a>. We also included factors with known roles at START (shown in red), which were not identified in our study. Among the G1 cyclins, we only included Cln3p, which is responsible for initiating the positive feedback loop of the large G1/S transcriptional program <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590-Skotheim1" target="_blank">[10]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590-Doncic1" target="_blank">[12]</a>. The other G1 cyclins, Cln1p and Cln2p, are important for this feedback, once it is initiated by Cln3p, but they were not included in this network. 60S ribosomal proteins are in yellow, while 40S ribosomal proteins are in orange. The most highly connected factors among the ones we identified are in green, and Cys4p is in blue.</p

Cell size correlates poorly with DNA content.

<p>We plotted the %G1 (x-axis) from all the deletion strains we examined against the haploid median cell size (in fl, y-axis) data of Jorgensen et al <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590-Jorgensen1" target="_blank">[23]</a>. The dashed lines indicate the cutoffs used in that study. We calculated and displayed the <i>r</i> values as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen-1002590-g003" target="_blank">Figure 3</a>. For every gene we included in this analysis, the values we used in this correlation are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002590#pgen.1002590.s001" target="_blank">Dataset S1</a>.</p