Sigenome smartpools library

Screening was performed in 384-well plates with a Dharmacon siGENOME SMARTpools library targeting all known protein kinases, phosphatases, tumour suppressor and DNA repair genes. The siRNA library was supplemented with non-targeting control siRNA, PLK1 siRNA as a viability control and CHK1 siRNA as a positive control. Briefly, cells were reverse transfected in triplicate at final siRNA concentration 20 nM. At 48 hours post transfection, cells were treated with 200 nM MK-1775 or vehicle, and survival was assessed after 96 hours exposure with CellTiter-Glo cell viability assay (Promega). CellTiter-Glo readings were first log2 transformed and then median centred. To assess the effect of siRNA on sensitivity to MK-1775 the log2 ratio between growth in MK-1775 plates and vehicle plates was assessed and expressed as a Z score, with the standard deviation estimated from the median absolute deviation (MAD). The MAD was calculated as the median of the absolute value of each value, x_i, minus the median: median(∣x_i - median(x_i)∣). Z-score of siRNA X = (cell viability effect of siRNA X – median cell viability effect of all 1206 SMARTpool siRNAs of the library) / MAD of all 1206 SMARTpool siRNAs of the library). A Z score of <−2, approximately the 95% confidence intervals, was considered evidence of increased sensitivity to MK-1775.

Screening was done in 384-well plates with a Dharmacon siGENOME SMART pools library targeting all known protein kinases and phosphatases, as described previously (5 (link)). To assess the effect of siRNA on growth/survival, the effect of siRNA in the vehicle plates was expressed as a Z score, with the standard deviation estimated from the median absolute deviation. To assess the effect of siRNA on sensitivity to PD173074, the log₂ ratio between growth in PD173074 plates and vehicle plates was assessed and expressed as a Z score.