Mk 2206

The AKT inhibitor MK-2206 (8-[4-(1-Aminocyclobutyl)phenyl)-9-phenyl-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3(2H)-one hydrochloride [1:1] was obtained from Cayman chemical (CAS 1032350-13-2). For experiments, cells were seeded at a density of 1.5 × 10⁵ in 6 cm dishes. The next day, MK2206 (diluted from a stock solution formulated in DMSO) was added to cultures at a concentration of 0.1 µM. After 24-hour exposure, the cells were collected and lysis for immunoblotting assays.

Culture media and supplements were purchased from Wisent (St-Jean-Baptiste, Quebec, Canada) and Gibco (Grand Island, NY, USA); AG1478, Gö6976, and MK-2206 were obtained from Cayman Chemical (Ann Arbor, MI, USA); LY294002 was purchased from Chemdea (Ridgewood, NJ, USA); rapamycin was obtained from LC Laboratories (Woburn, MA, USA); and rhEGF was a gift from Dr. Stéphane Lefrançois (INRS—Centre AFSB, Laval, QC, Canada).

The primers were synthesized and PAGE purified by IDT DNA. Fugene 6 transfection reagent was purchased from Promega while Lipofectamine RNAiMax was from Life Technologies/Invitrogen. All cell culture reagents including media, antibiotics were from GIBCO/Invitrogen. Antibodies to total Ubiquitin (clone P4D1) was from Santacruz Biotechnology, Luciferase (Millipore), pAKT, AKT (Cell Signaling), Ubiquitin, Lys63 specific (clone Apu3, Millipore), His-tag (clone H3 and C-term, Invitrogen) or Millipore (H8 clone). FLAG tag-HRP antibody (Clone M2) was purchased from Sigma Aldrich. HRP-conjugated and fluorophore-conjugated secondary antibodies were from Jackson ImmunoResearch. ON-TARGET plus siRNA to TRAF6 and scrambled control siRNA were obtained from GE Life Sciences/Dharmacon. D-Luciferin was from Xenogen Corp while GloSensor was from Promega. Erlotinib was a kind gift from Genentech. Tyrphostin AG1478 was obtained from Cayman Chemical. TNF-α antagonist (WP9QY) and IL-1R antagonist were from SantaCruz biotechnology. Recombinant IGF-1, IL-1α were from PepreoTech. CI-1040, GF109203X, NVP-AEW541 and MK2206 (Cayman Chemical), and Linsitinib (LC Laboratories). Protein A and Protein G sepharose beads were from GE HealthCare. Purified NEDD4-1 (Sigma Aldrich), Myc-tagged ubiquitin (Cat. No. U-115) UBE1 (Cat. No. E305), and UBCH5 (Cat. No. E2-616) all were from Boston Biochemicals.

BMDMφs and PMφs were cultured for 24 h with human medium oxLDL (100 μg/ml, Kalen Biomedical, no. 770202) or cholesterol (50 μg/ml, Sigma-Aldrich, no. C3045), followed by ultrapure LPS stimulation (10 ng/ml, InvivoGen, no. tlrl-3pelps) for up to 6 h, or CpG (300 nM) (InvivoGen, no. tlrl-1826), Pam₃CSK₄ (10 ng/ml) (Tocris Bioscience, no. 4633), or bpV (HOpic) (10 µM) (Sigma-Aldrich, no. SML0884) stimulation for up to 3 h. Ethanol (0.5%) was used as a carrier control for cholesterol. For inhibitor experiments, MK-2206 (3 µM) (Cayman Chemical, no. 11593), AKT2i (3 µM) (Sigma-Aldrich, no. 124029), Mito-TEMPO hydrate (25 µM) (Cayman Chemical, no. 16621), and bpV (HOpic) (10 µM) were added 1 h prior to LPS stimulation. For assessing the accumulation of oxLDL, a mixture of DiI-oxLDL and oxLDL (10 and 90 μg/ml, respectively) were added to PMφs for 24 h. In all experiments, Mφs were first cultured for 24 h with or without oxLDL or cholesterol. The groups without oxLDL or cholesterol served as controls. The control group for cholesterol loading was cultured with media containing the same concentration of ethanol as the cholesterol group. Cells were then stimulated with LPS from 0 up to 3 h. At each time point, the groups with versus without oxLDL or cholesterol were compared and changes over time were determined.

PC3 parental, CKB shRNA or knockout cells were treated with DMSO (#276855, Sigma) or 25uM MK-2206 (#11593, Cayman Chemical) for 48h before collecting cell lysate. For cell proliferation assay, 600 cells were seeded into 96-well plate in RPMI 1640 culture medium containing 10% FBS. In the next day, medium was changed to RPMI 1640 supplemented with 1.25% FBS. Fluorescence was detected every 24 h using Alamar Blue reagent, incubating at 37°C for 4 h, then reading on a fluorescence plate reader (#Infinite M1000, Tecan) with excitation at 530nm and emission at 595nm.