Drug combination using dasatinib and CX-4945.

<p><b>A.</b> The Chou-Talalay method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144126#pone.0144126.ref078" target="_blank">78</a>] was used to perform drug combination studies of dasatinib and CX-4945. The points represent the average viability ± standard error of mean following 72 h of drug treatment at the indicated concentrations of CX-4945 (•) and CX-4945 + dasatinib (◆; constant molar ratio of 20:1 of CX-4945:dasatinib) for the various EOC cell lines as a percentage of vehicle treated cells. The curve-fit lines were generated using non-linear regression analysis in GraphPad Prism. Data for the other molar ratios that were evaluated are presented in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144126#pone.0144126.s002" target="_blank">S2 Fig</a>. B.</b> The dose response data were used to calculate the Combination Index (CI) values for each cell line at the various molar ratios using CalcuSyn software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144126#pone.0144126.ref079" target="_blank">79</a>]. CI values less than 1 suggest that the drugs are working synergistically. Shown is the average calculated CI value ± standard error of the mean.</p

Gene expression in clinical samples.

<p>Agilent gene expression data from TCGA on 518 serous cystadenocarcinomas and 8 fallopian tube samples derived from healthy individuals were queried for 29 dasatinib sensitizing genes. The six Agilent probes that showed ≥ 1.5-fold increase in the average gene expression of the respective genes in the tumor samples (gray boxes) relative to the controls (white boxes) are shown. The whiskers of each box plot represent the expression values at the 5^th and the 95^th percentiles. The p-values were calculated using an unpaired two-tailed t-test using GraphPad Prism. <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144126#pone.0144126.s007" target="_blank">S4 Table</a></b> lists the average expression values of the Agilent probes across the tumor and normal samples for all 29 genes.</p

Quantification of cell cycle and apoptosis assays.

<p>Cell cycle and apoptosis data were quantified for the indicated fold-changes relative to vehicle treated cells and are presented as bar graphs showing the average fold-change ± standard error of mean. In all three assays, single (das, 0.5 μM; CX-4945, 10 μM) and combination drug treatments (das, 0.5 μM; CX4945, 10 μM) were for 72 h. P-values were calculated using a t-test comparing the combination treatment group to each single agent treatment group. The dashed line indicates the theoretical value if the drugs act additively calculated using the Bliss independence model (Bliss additivity value = FC_Das + (FC_CX-4945 * (100—FC_Das))/100 where FC is fold-change [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144126#pone.0144126.ref051" target="_blank">51</a>]. Observed values larger than the Bliss additivity value indicate synergy. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144126#sec010" target="_blank">Materials and Methods</a> for additional assay details.</p

Overview of the design and work flow of experiments.

<p><b>A</b>-<b>F.</b> A general schematic of the experimental workflow of the primary and secondary siRNA screening and subsequent validation and refinement experiments performed to identify the second-site sensitizers for dasatinib. Details for each set of experiments are provided in the subsequent Figures and Supplementary Figures and Tables throughout the Results section.</p

Correlation of gene expression to dasatinib sensitivity.

<p><b>A.</b> The basal level of gene expression of 29 dasatinib-sensitizing genes in seven EOC cell lines was measured by using quantitative PCR performed with a 96☓96 dynamic array on the Fluidigm BioMark microfluidic platform. Shown is a representative heat map of the dynamic array. Delta C_t values were calculated for each gene in each cell line (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144126#sec010" target="_blank">Materials and Methods</a> for details). <b>B.</b> Data on the dose response to dasatinib for seven EOC cell lines were generated and cell viability at 1 μM dasatinib was calculated for each cell line as a percentage of vehicle treated cells using GraphPad Prism. Shown is the average ± standard error of mean for each data point. <b>C.</b> Delta C_t and dasatinib sensitivity data (i.e. viability at 1 μM drug concentration) were subjected to Spearman Correlation analysis using GraphPad Prism. The magnitude of correlation (Spearman r value) is shown for the four genes which showed a statistically significant correlation (p < 0.05). Each point represents an EOC cell line with the color matching the code shown in panel 2B. The line through the data points is for illustrative purposes only. <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144126#pone.0144126.s006" target="_blank">S3 Table</a></b> lists the r and p-values for the other genes evaluated but which did not show significance.</p