Ipatasertib gdc 0068

Experiments were performed mostly using H1299 cells (non-small-cell lung carcinoma human cell line) [NCI-H1299 (ATCC® CRL5803™)], unless stated otherwise. Other cell lines used were MDM2/MDMX double KO H1299 cell line (44 (link)), A549 cells (Adenocarcinomic human alveolar basal cell line), U2OS cells (Human bone osteosarcoma epithelial Cell line), SAOS-2 cells (Human bone osteosarcoma cell line), A375 (p53WT), A375 (p53KO) (Human melanoma cell line) and Raji cells (type III latent Burkitt's Lymphoma). Cell lines were cultured in RPMI 1640 medium or in DMEM–Dulbecco's Modified Eagle Medium (for U2OS, A375 and A549 cell lines) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (Invitrogen) and 5 μg/ml Plasmocin prophylactic (Invivogen). Cell lines were routinely checked for mycoplasma contamination using PlasmoTest kit (Invivogen). Drugs: MG132 (474790-5, Calbiochem), PI3Kδ inhibitor CAL-101 (S2226, Selleck Chemicals), PI-3065 (S7623, Selleck Chemicals), AKT kinase inhibitor Ipatasertib (GDC-0068) (SES22808, Selleck Chemicals), Doxorubicin (Sigma-Aldrich), Cycloheximide (C4859, Sigma-Aldrich).

The TNBC cell line MDA-MB-231 was purchased by ATCC (Rockville, USA) and their brain-seeking subclone MDA-MB-231BR was a kind gift from Prof. Dr. Wikman-Kocher (Institute of Tumor Biology, University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany). All cell lines were cultured in DMEM-medium (Gibco, ThermoFisher Scientific, Waltham, MA USA) supplemented with 10% (v/v) fetal calf serum (FCS) under standard conditions in a water-saturated atmosphere containing 5% CO₂ at 37°C. Ipatasertib (GDC-0068) was purchased from Selleckchem (Cat.-No. S2808, Biozol, Eching, Germany, dissolved in Dimethylsulfoxide (DMSO)) and used in concentrations of 1 to 20 µM.