Akt inhibitor mk 2206

The human GC cell lines MGC803, BGC823, MKN-45, SGC7901, AGS, and the immortalized gastric epithelium cell line (GES-1) were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Primary GC cell line XN0422 was established in our laboratory^{55 (link)}. Cell culture was conducted as previously described^{56 (link)}. The cells were treated as follows: PI3K inhibitor LY294002 (CST) 10 μM for 24 h; AKT inhibitor MK2206 (Selleck Chemicals) 5 μM for 24 h; NF-κB inhibitor JSH-23 (Abcam) 10 μM for 24 h; IKK inhibitor BAY 11–7082 (Abcam) 10 μM for 12 h; NF-κB activator TNF-α (Sigma-Aldrich, USA) 10 ng/mL for 24 h.

For inhibitor studies the following reagent were used: The glycolysis inhibitors 3-bromopyruvate (Sigma-Aldrich, St. Louis, MO, USA) and L-glyceraldehyde (Sigma-Aldrich, St. Louis, MO, USA) solved in PBS were added from a sterile stock solution to final concentration of 0.25 mM and 1 mM, respectively. SR13800 (Tocris, Bristol, United Kingdom) and AZD3965 (Selleck Chemicals, Houston, TX, USA) - inhibitors of MCT1 -solved in DMSO were added from a sterile stock solution to final concentration of 0.1 µM and 10 µM, respectively. AKT inhibitor MK-2206 (Selleck Chemicals, Houston, TX, USA) solved in DMSO were added from a sterile stock solution to final concentration of 1 µM. mTOR inhibitors Rapamycin (Calbiochem, Billerica, MA, USA) and OSI027 (Selleck Chemicals, Houston, TX, USA) solved in DMSO were added from a sterile stock solution to final concentration of 10 µM. AMPK inhibitor Dorsomorphin and AMPK activator AICAR (both Selleck Chemicals, Houston, TX, USA) solved in DMSO were added from a sterile stock solution to final concentration of 1 µM or 1 mM, respectively. As control, inhibitor solvent was used.

Renal fibroblasts were stimulated with TMAO (300 µM, Sigma-Aldrich, St. Louis, MO, USA), TNF-α (1, 10 or 50 ng/ml, Sigma-Aldrich) or the combination of both for 24-96 h, depending on the experimental setup, at 37 °C in 5% CO₂. Renal fibroblasts were pre-stimulated with TMAO for 2 h prior to TNF-α stimulation during the combination treatments. The renal fibroblasts were also pre-incubated with DMSO (vehicle), mTOR inhibitor ridaforolimus (1 µM, Selleckchem, TX, USA), Akt inhibitor MK-2206 (1 µM, Selleckchem), PI3K inhibitor wortmannin (1 µM, Selleckchem) or ERK inhibitor PD98059 (10 µM, Santa Cruz Biotechnology Inc., Heidelberg, Germany) for 1 h prior to TMAO or TNF-α stimulation. Supernatants were collected and stored at -80ºC until further investigation.

The antibodies for Western blot analyses of P-YB-1 (S102) (#2900), YB-1 (#4202), P-Akt (S473) (#4060), GAPDH (#2118S), cleaved PARP (#9541S), P-p53 (S20) (#9287P), p53 (#9282S) and p21 (#2947S) were purchased from Cell Signaling Technology (Frankfurt, Germany). The Akt antibody (#610877) was purchased from BD Biosciences (Heidelberg, Germany). The KRAS antibody (#ab157255) and the antibody for immunofluorescence staining of P-YB-1 (#ab47162) were purchased from Abcam (Cambridge, UK). The actin antibody (#A2066) was purchased from Sigma-Aldrich (Taufkirchen, Germany). The anti α-tubulin antibody (cat. #CP06) was purchased from Calbiochem (Schwalbach, Germany). Anti-Ki67 (#A2066) was purchased from Agilent (Waldbronn, Germany). The Human Phospho-MAPK array kit was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Anti-phospho-Histone H2AX (Ser139) antibody (#05-636) was purchased from Merck Millipore (Darmstadt, Germany). The RSK inhibitor LJI308 (#S7871) and Akt inhibitor MK2206 (#S1078) were purchased from Selleckchem (Munich, Germany). 5-fluorouracil (#84300-VO) was purchased from Medac (Hamburg, Germany). Cetuximab was provided by the pharmacy at the University of Tuebingen (Tuebingen, Germany). Doxycycline (#A2951) was purchased from AppliChem (Darmstadt, Germany).

Renal fibroblasts were stimulated with TMAO (100 µM, 200 µM, or 300 µM; Sigma-Aldrich) for 24–96 h, depending on the experimental setup, at 37 °C in 5% CO₂. As a positive control, renal fibroblasts were stimulated with TGF-β1 (10 ng/mL Invivogen, CA, USA). The renal fibroblasts were also pre-incubated with DMSO (vehicle), PERK inhibitor GSK2656157 (0.5 µM, Santa Cruz Biotechnology Inc., Heidelberg, Germany), Akt inhibitor MK-2206 (1 µM, Selleckchem, Houston, TX, USA), mTOR inhibitor ridaforolimus (1 µM, Selleckchem), and PI3K inhibitor wortmannin (1 µM, Selleckchem) for 1 h prior to TMAO stimulation. Supernatants were collected and kept at −80 °C until further analysis.

CGNs and/or CNs were prepared7 (link)8 (link) and seeded at 4 × 10⁵ cells per ml in 96- or 24-well plates with neuronal media for 3 and 21 days, respectively. T-cell lines were re-stimulated with antigen and APCs for 48 h and are referred to as activated T_enc cells. Neurons and activated syngeneic T_enc cells were washed twice and co-cultured at a 1:1 ratio for 24 h, unless stated otherwise. MOG_35–55 T-cell line and MBP_89–101T-cell line were co-cultured with neurons from C57BL6 mice and C57BL/10.RIII mice, respectively.
For some experiments, PI3K inhibitor Wortmannin (catalogue number S2758, Selleckchem), Akt inhibitor MK-2206 (catalogue number S1078, Selleckchem), rIFNβ (12405-1, PBL Assay Science) and anti-PDL1 (10 μg ml⁻¹, Anti-Mouse CD274/B7-H1) Functional Grade Purified, 16-5982-82, eBioscience) were added to the CGNs. Anti-PD1 (10 μg ml⁻¹, Anti-Mouse CD279/PD-1, 16-9985-82, eBioscience) was added to T cells before co-cultures.

The human immortalized normal hepatic cell line LO2 and HCC cell lines (PLC/PRF/5, SNU-475, SK-Hep-1, Hep3B and Huh7) were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco, GrandIsland, NY, USA) at 37°C with 5% CO2. Akt inhibitor MK-2206 (1 μM, Selleck Chemicals, Houston, TX, USA), 5-aza-2′-deoxycytidine (5-Aza-dC) (Sigma-Aldrich, St Louis, MO, USA) was used to treat HCC cells following the manufacturer's instructions.