Ly294002 ly

Tyrphostin AG1478 (AG) was purchased from Biomol Research Laboratories (Milan, Italy). PD98059 (PD) and Sirtinol were obtained from Calbiochem (Milan, Italy). 1-[4-(-6-Bromobenzol1,3diodo-5-yl)-3a,4,5,9btetrahydro-3H-cyclopenta[c−] quinolin8yl] ethanone (G-1) was purchased from Tocris Bioscience (Bristol, UK). E2, H89, LY294002 (LY) and ETO were purchased from Sigma-Aldrich Srl (Milan, Italy). All compounds were solubilized in dimethyl sulfoxide (DMSO), except E2 and PD which were dissolved in ethanol.

A feeder-free adapted mES line R1 was propagated as described previously [29 (link)] in an undifferentiated state by cell culturing on tissue culture plastic coated by gelatin (0.1% porcine gelatin solution in water) in Dulbecco's modified Eagle's medium (DMEM) containing 15% fetal bovine serum (FBS), 100 IU/mL penicillin, 0.1 mg/mL streptomycin, 1x nonessential amino acid (all from Gibco-Invitrogen, UK), and 0.05 mM β-mercaptoethanol (Sigma-Aldrich, USA), supplemented with 5 ng/mL of leukemia inhibitory factor (LIF, Chemicon, USA) referred to here as the complete medium.
APO, DPI, hydrogen peroxide (H₂O₂), N-acetylcysteine (NAC), and LY294002 (LY) were provided from Sigma-Aldrich. Stock solutions of APO (0.4 M), DPI (10 mM), and LY (10 mM) were prepared by dissolving the compounds in dimethyl sulfoxide. Aliquots were stored at −20°C. NAC was prepared as a 0.5 M stock solution in serum-free DMEM medium, pH was adjusted to 7.4, and filter-sterilized aliquots were stored at −20°C. Drugs were added directly to the incubation medium or freshly prediluted in sterile phosphate-buffered saline (PBS) to desired concentration.

Anti-MMP-9 polyclonal goat antibody, anti-β-actin monoclonal mouse antibody, goat anti-rabbit IgG/HRP, goat anti-mouse IgG/HRP, rabbit anti-goat IgG/HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Phospho-EGFR rabbit mAb, anti-EGFR rabbit mAb, anti-p44/42 MAP Kinase monoclonal rabbit antibody, anti-Phospho-p44/42 MAP Kinase monoclonal rabbit antibody, anti-AKT monoclonal rabbit antibody, anti-Phospho-AKT monoclonal mouse antibody, anti-Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit mAb, anti-SAPK/JNK rabbit mAb, anti-Phospho-p38 MAPK (Thr180/Tyr182) (12F8) Rabbit mAb and anti-p38 MAPK rabbit antibody were purchased from Cell Signaling Laboratories (Beverly, MA). PMA (Phorbol-12-myristate-13-acetate), specific inhibitors of PI3K [LY294002 (LY)], MAPK family [PD98059 (PD), mitogen-activated protein kinase kinase (MEK) inhibitor; SP600125 (SP), c-jun N-terminal kinase (JNK) inhibitor and SB203580 (SB), P38 MAPK inhibitor], gelatin and FITC-phalloidin were purchased from Sigma Chemical Co, (St.Louis, MO). PC, cholesterol and PEG4000 were purchased from Sigma Chemical Co, (St.Louis, MO); Honokiol was separated and purified by our laboratory and its purity and structure were analyzed and identified by high performance liquid chromatography and nuclear magnetic resonance [16 ].

Human NB tumor cell lines NB1, NB3, SH-SY5Y, and IMR32, kindly provided by Dr Luca Longo, from IRCCS AOU San Martino – IST, Genoa, Italy, were cultured in RPMI-1640 medium (Sigma-Aldrich, Milan, Italy) with addition of antibiotics (1%), and fetal bovine serum (FBS; 10%; Gibco, Life Technologies, Monza, Italy). DNA typing for the cell line authentication was done prior the analyses. For each experiment, equal numbers of cells were seeded, and the concentration of DMSO equivalent to those of the chemicals used, was applied to control cells. Entrectinib was provided by Ignyta Inc. (San Diego, CA) under a material transfer agreement; Chloroquine (CQ), 3-Methyladenine (3-MA), Tamoxifen (Tam), Rapamycin (Rapa), Ly294002 (Ly), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich, crizotinib from Selleck Chemicals (Munich, Germany). The concentrations of the compounds used in the study were adapted for each experiment before use.

Genistein (Sigma Chemical Co., St. Louis, MO, USA) was dissolved in 0.1 M Na₂CO₃ to create a 10-mM stock solution. TPA (Sigma-Aldrich, St. Louis, MO) was prepared in phosphate-buffered saline (PBS; 137 mM NaCl, 1.4 mM KH2PO4, 4.3 mM Na2HPO4, 2.7 mM KCl, pH 7.2). For analysis of the signaling pathways involved in TPA-induced DNA-binding of AP-1 and NF-κB, we also treated HepG2 cells with the p38 inhibitor SB203580 (SB), the MEK/ERK inhibitor PD98059 (PD), the JNK inhibitor JNKI, the IKK inhibitor BMS (AKTI), LY294002 (LY, an Akt inhibitor) and bisindolylmaleimide (GF, GF109203X, a PKC inhibitor) were purchased from Sigma-Aldrich to block these pathways.