Akt inhibitor 8

CCK-8 assay was used for cell proliferation and drug susceptibility tests. Drugs used were AKT inhibitor VIII (HY-10355), gefitinib (HY-50895) and AZD-0530 (HY-10234), were purchased from MedChem Express (Monmouth Junction, NJ, USA). The cells were seeded in 96-well plates (800 or 5000 cells/well) and cultured for 4 days or 3 days. Cell viability was evaluated using the Cell Counting Kit 8 (CCK8; Dojindo, Japan) according to the manufacturer’s instructions. Briefly, 10 µL CCK-8 solution was added to each well at pre-determined time points, and the cells were incubated for 2 h. Absorbance at 450 nm was detected using a microplate reader (Bio-Rad, USA).

OnTarget plus siRNA smart pools were from Dharmacon/Horizon Discovery, Waterbeach, UK. Individual mice cytokines were purchased from Immunotools, Friesoythe, Germany, and LPS was purchased from Invitrogen/Thermo Fischer Scientific, Waltham, MA, USA. Propidium iodide was from Calbiochem/Merck, Darmstadt, Germany, and Hoechst33342 from Thermo Fischer Scientific, Waltham, MA, USA. Rapamycin was bought from Hycult Biotech, Uden, Netherlands, AKT inhibitor VIII from MedChem Express, Monmouth Junction, NJ, USA and cycloheximide from Merck, Darmstad, Germany. L-Azido-homo-alanine (L-AHA) and Protein synthesis detection Kit were from JenaBioscience, Jena, Germany. Real-time Glo apoptosis/necrosis kit was purchased from Promega, Fitchburg, USA. Insulin, holo-transferrin, sodium selenite and dexamethasone were from Sigma-Aldrich/Merck, Darmstadt, Germany. Lipofectamine RNAimax reagent was purchased from Invitrogen. BCA assay kit was from Serva Electrophoresis GmbH. ECL-substrate solution was from Thermo Fischer Scientific. DAPI-containing fluoroshield mounting medium was from Sigma-Aldrich/Merck, Darmstadt, Germany. Cyto ID autophagy kit was purchased from Enzo life sciences, Lörrach, Germany.