DNA Content Analysis and Drug Interactions Figure 4. Gemfibrozil delays initiation of DNA replication. A, The critical cell size (shown in fl) of diploid BY4743 cells treated with DMSO, rapamycin (0.1 mg/ml) or gemfibrozil (50 mg/ml), was measured from synchronous elutriated cultures, in YPD medium. The data points shown were from three independent experiments in each case. The P values shown were calculated from paired, two-tailed t tests, assuming unequal variance. The data used to calculate these parameters are shown in Figure S1. B, The specific rate of cell size increase constant k (in h21) was measured from the same elutriation experiments shown in a, assuming exponential growth. The data used to calculate these parameters are shown in Figure S1. C, The cell size distributions of the indicated cell populations, proliferating asynchronously in YPD medium, were measured using a channelyzer (see Materials and Methods, and [22]). 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 [22]. doi:10.1371/journal.pone.0036503.g004 distribution was recorded. We used the Accucomp Beckman software package to obtain the statistics of each size distribution. Measurements of critical size and growth rate from elutriated cultures For isolation of early G1 daughter cells, cultures were grown in YPD at 30uC to a density of ,1–56107 cells/ml, then fractionated with a Beckman JE-5.0 elutriator as described previously [16]. Early fractions containing predominantly (.95%) small unbudded cells were collected by centrifugation, re-suspended in fresh medium and aliquoted in three separate flasks. To each flask, we then added as indicated rapamycin (at 0.1 mg/ml), gemfibrozil (at 50 mg/ml), or DMSO alone. After testing several doses of each drug and measuring the DNA content, we decided to use these concentrations because they were the lowest ones that resulted in consistently pronounced effects in this strain background. The cultures were incubated at 30uC. Every 20 min we monitored the percentage of budded cells and cell size. The ‘‘critical size’’ is the size at which 50% of the cells have budded in these experiments, and it was calculated as we described elsewhere [16]. To calculate ‘‘growth rate’’ assuming exponential growth, we plotted the natural log (ln) of cell size as a function of time (in h), see Figure S1. We fit the data to a straight line using the regression function in Microsoft Excel. From the slope of the line, we obtained the specific rate of cell size increase constant (k, in h21). The average of all experiments (n = 3) for each treatment was then calculated, along with the associated standard deviation. Staining for DNA content analyses Fixed cells were stored at 4uC overnight to 14 days. Cells were collected by centrifugation and stained overnight in 0.5 ml staining solution containing 50 mM sodium citrate pH 7.0, PLoS ONE | www.plosone.org 6 May 2012 | Volume 7 | Issue 5 | e36503