Detection was done utilizing HRP conjugated secondary antibodies and enhanced chemiluminescence reagent. Cell tradition Cell lines were sourced as explained previously and were cultured in RPMI 1640 medium supplemented with 2 mM L glutamine, 10 % FBS, 100 units/ml penicillin and 0. 1 mg/ml streptomycin at 37 C in 5%CO2. Cells were treated with inhibitors reversible Chk inhibitor diluted in DMSO as described in the Figure legends. Just before lysis, cells were then lysed on ice and washed with PBS. Lysates were snap frozen in liquid nitrogen, centrifuged at 18000 g for 15 min at 4 C and supernatants were stored at?80 C. For transient transfections of HEK 293 cells, cells cultured on 10 cm diameter dishes were transfected with 5 g of the indicated plasmids using polyethylenimine. Cell growth and invasion assays Cells were seeded into the interior 60 wells of Organism 96 well plates in triplicate and permitted to attach overnight. For inhibitor solutions, cells were treated with 10 nM 10 M MK 2206, AZD5363 or AZD8055 diluted in DMSO. At 72 h later cell viability was determined using CellTiter 96 AQueous One Solution Cell Proliferation Assay based on the manufacturers directions. Results were plotted with a best-fit sigmoidal variable mountain dose response curve and GI50 values were determined using GraphPad Prism 5. 0. Chest cell line screen testing for AZD5363 was performed as described previously. The ability for proliferation following SGK1 knock-down was dependant on seeding 2000 cells/well to the interior 60 wells of 96 well plates 48 h post transduction with lentiviral shRNAs. The MTS analysis was then performed 24, 48, 72, 96, 120 and 144 h article seeding. Results are presented while the change in absorbance over the 5-day period in accordance with the assay start point. The cells were assayed in triplicate. The power of BT 549 cells to invade was tried in a growthfactor paid down MatrigelTM c-Met inhibitor attack chamber based on the manufacturers directions. Briefly, cells were serum deprived for 2 h, detached using a celldissociation buffer and 2. 5 105 cells stopped inRPMI 1640 medium containing 10 percent BSA were added to the top of chambers in triplicate and chemoattractant was added to the lower wells. The chambers were kept at 37 C in five full minutes CO2 for 20 h. Cells that did not invade were taken off top of the face of the filters and cells that had moved to the reduced face of the filters were fixed and stained with Reastain Quick Diff kit and images were captured. For cell attack assays, statistical significance was assessed by one-way ANOVA followed by Tukeys multiple comparison test using GraphPad Prism 5. 0. shRNA mediated SGK1 knock-down using a lentiviral supply system To knock down SGK1 we applied the MISSIONTM shRNA system obtained from Sigma Aldrich.