9 research outputs found
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
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<sub>Das</sub> + (FC<sub>CX-4945</sub> * (100—FC<sub>Das</sub>))/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
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<sub>t</sub> 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<sub>t</sub> 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
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<sup>th</sup> and the 95<sup>th</sup> 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
Hits unique to the EOC cell lines.
<p><b>A.</b> A heat map representation of the viability scores for eight EOC and three HIO cell lines achieved from secondary screens of the siRNA library targeting 300 genes. All viability score values range between 0.12 and 1.53. Shades of green represent reduced viability (<0.80), shades of red represents increased viability (>0.80), and black represents a viability score of 0.80. The heat map was generated using MultiExperiment Viewer. Floral diagrams in yellow and blue show the number of hits across either the eight EOC or three HIO cell lines, respectively, and the intersection of the hits within each group. <b>B.</b> A Venn diagram shows the number of hits in common between the HIOs and EOCs and the number hits unique to each group.</p
Validation of hits via siRNA deconvolution screening, qRT-PCR and Western blotting.
<p><b>A.</b> The seven hits selected for further studies were validated by performing deconvolution screens where individual siRNAs which were initially a part of a pool of four siRNAs in the secondary screens were evaluated for their effects on cell viability. For each hit being validated, the average normalized viability scores (±SD) following gene silencing using each of the four species of siRNAs evaluated in the eight EOC cell lines are shown. Hits were considered on-target if viability scores of less than 0.85 were observed for all eight EOC cell lines for at least two independent siRNA species targeting a gene. The bar graphs represent the eight EOC cell lines in the following order: A1847, A2780, C30, CP70, OVCAR5, OVCAR8, SKOV3, and UPN275. <b>B.</b> The two most effective siRNAs targeting each gene were pooled (12.5 nM each siRNA species) and the effect on cell viability was quantified. The bars represent the eight EOC cells lines as described in Panel A. <b>C.</b> qRT-PCR was performed on all of the eight EOC cell lines following gene silencing for 72 h using a pool of the two most effective siRNAs identified from the deconvolution studies from panel A for the four hits that were determined to be on target. The asterisk represents <i>PTN</i> mRNA levels which are below the level of detection following siRNA treatment (i.e. complete knockdown of mRNA). <b>D.</b> Western blot analysis was performed following gene silencing for either 72 h (<i>HSPA5</i>, <i>NDC80</i>, and <i>PTN</i>) or 120 h (<i>NUF2</i>) to determine the level of knockdown at the protein level in A1847 cells. Immuno blots were quantified using AlphaView software, version 3.3 (Cell Biosciences).</p
Assessment of gene expression using TCGA ovarian cancer data set.
<p>TCGA data set on 494 ovarian serous adenocarcinomas was queried to determine the mRNA expression levels (log<sub>2</sub>(tumor/normal ratio)) of <i>NDC80</i>, <i>NUF2</i>, <i>PTN,</i> and <i>HSPA5</i>. These data are shown as bar graphs with the grey dashed lines indicating the percentage of samples with 1.5-fold, 3-fold, 5-fold and 10-fold overexpression as compared to unmatched normal samples in the TCGA data set.</p
Assessment of clinical significance using an independent cohort.
<p><b>A.</b> Shown are the gene expression levels for 53 serous adenocarcinomas normalized to the mean gene expression levels measured in normal ovarian tissue (n = 10). <b>B.</b> Shown are the mean gene expression levels across the serous adenocarcinomas for <i>NDC80</i>, <i>NUF2, PTN</i> and <i>HSPA5.</i> A two-tailed t-test indicates that the increase in gene expression in the tumor samples is statistically significant relative to the normal tissue. *** = p<0.0005; ** = p<0.005; * = p<0.05.</p
List of hits validated following deconvolution of siRNA pools.
<p>List of hits validated following deconvolution of siRNA pools.</p