Fred Hutchinson Cancer Research Center (FHCRC1): Functional Landscape of the Human Kinome in MYCN Amplified and Non-amplified Neuroblastoma
In order to identify candidate drugs targets that exhibit lethality only in the context of MYCN amplification, we carried out a set of siRNA screens focused on the kinome, targeting ~713 kinases, utilizing human neuroblastoma cells lines with or without MYCN amplification. The neuroblastoma cell lines were: SK-N-BE2 (MYCN amplified) and SK-N-AS (non-amplified). The kinase Hits for the MYCN amplified cell line were selected using a combination of their differential activity when compared to the non-MYCN amplified cells and also ranked by P-values, based on the replicates.
Neuroblastoma cell lines, SK-N-BE2 (MYCN-amplified) and SK-N-AS (non-amplified) were obtained from the American Tissue Type Collection (ATCC) and cultured in RPMI with 10% FBS and Penicillin and Streptomycin at the concentration recommended. 25mM HEPES was added during the screens to minimize variation of pH during handling of the plates.
High-throughput siRNA transfections
Transfection feasibility for each cell line was established using a factorial-based optimization scheme with variable parameters in cell density, liposomal, and siRNA concentration. All transfection conditions were statistically evaluated using a simple Z-factor score to determine differentials between mock-transfected cells (transfection reagent only) versus a positive control for growth inhibition (KIF11) and a negative control (non-targeting siRNA). Based on these preliminary experiments performed on each cell line, the optimal transfection condition selected for each cell line was: 1)SK-N-BE2, cells were seeded at 1600 cells/well in 384 well. The next day 5 μl of Dharmafect I (18.75 μl/ml) and siRNA (2.5 pM/well) mix was added and viability with CellTiter-Glo was read at 96 hr after plating; 2)SK-N-AS: seeded at 2000 cells/well and the next day 5 μl of Dharmafect I (18.75 μl/ml) and siRNA (2.5 pM/well) mix was added and viability with CellTiter-Glo was read at 96 hr after plating. The siRNA library targeting the human kinome (~713 kinases) was tested in triplicates to establish experimental variability and statistical validity. A detailed description of the method is available in the supplementary information by Toyoshima et al [Toyoshima, 2012 #4918].
The siRNA library targeting 713 human (MISSION® siRNA Human Gene Family Set, Sigma) was tested with viability as the phenotypic endpoint. Cell viability was quantified using an Envision Multilabel detector/plate reader (PerkinElmer) with the CellTiter-Glo assay (Promega) that measures ATP concentrations in cell lysates. siRNA libraries utilized pools of 3 independent siRNAs targeting each gene, in a one gene per well approach. RNAi screens were performed in 384-well format in triplicate, in independent plates, utilizing robotics instrumentation available at the University of Washington - Quellos facility.
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