Figure 1.
PARP-inhibitor synthetic lethality screen with protein kinase siRNA library. (A) HTS method. CAL51 cells plated in 96-well plates were transfected with siRNA. Each transfection plate contained 80 experimental siRNAs (SMARTPools of four different siRNA targeting the same gene) supplemented with four wells of non-targeting siCON, and two wells of siRNA directed against BRCA1 (positive control). Transfected cells were divided into six replica plates, half treated with DMSO vehicle alone and half with PARP inhibitor KU0058948 at 1 μM, the SF80 of CAL51. Cell viability was assessed after 5 days of KU0058948 exposure using CellTiter-Glo Luminescent Cell Viability Assay (Promega). (B) Reproducibility of HTS method. Correlation of the effect of siRNA on cell growth in vehicle-treated plates from two replicates of the entire screen. Spearman correlation coefficient, r=0.83. (C) Correlation of KU0058948 sensitivity Z-scores from two replicates of the entire screen. r=0.54. (D) Scatter plot of averaged Z-scores from PARP inhibitor sensitivity screen carried out in duplicate with KU0058948. Red line indicates −3 averaged Z-score significance threshold. Black, siRNA (SMARTPools) targeting 779 protein kinase genes; red, siCON and blue, siBRCA1. Reflecting the reproducibility and sensitivity of the screen, siCON and siBRCA1 Z-scores were widely separated, with a screen Z′-factor (Zhang et al, 1999) of 0.34.