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).