Ar a014418

The reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA), including SNAP (a soluble donor of NO [35 (link)]), AR-A014418 (a selective GSK-3β inhibitor [41 (link)]), and pyrrolidine dithiocarbamate (PDTC; an NF-κB inhibitor [43 (link)]). Neurobasal (NB) A medium, Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) and fetal bovine serum (FBS) were obtained from Gibco (Life Technologies, CA, USA). Monoclonal or polyclonal antibodies against NF-κB p65 (Cat# ab16502), phosphorylated p65 (Ser536; Cat# ab86299), GSK-3β (Cat# ab93926), phosphorylated GSK-3β (Ser9; Cat# ab131097), and Tju1 (neuron-specific class III beta-tubulin; Cat# ab78078 and ab18207) were from Abcam (Cambridge, CA, USA). Mouse monoclonal antibody against CGRP (Cat# sc-57053) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Protein kinase B (Akt1; Cat# 2938) and phosphorylated Akt1 (Ser473; Cat# #4060) were from Cell Signaling Technology (Beverly, MA, USA). Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 555-conjugated goat anti-mouse IgG were provided by Invitrogen (Carlsbad, CA, USA). The FastStart Universal SYBR Green Master kit (Cat# 04913850001) and the first-strand cDNA synthesis kit (Cat# 04896866001) were purchased from Roche (Roche Applied Science, Germany).

Human renal carcinoma (Caki-1 and A498), human lung cancer (A549), and human breast cancer (MDA-MB361) were procured from American Type Culture Collection (Manassas, VA, USA). Human recombinant TRAIL, zVAD-fmk, and anti-survivin were provided by the R&D system (Minneapolis, MN, USA). MG132, PD98059, AG-490, compound C, and NVP-BEZ235 were supplied from Calbiochem (San Diego, CA, USA). Dexamethasone, cycloheximide, AR-A014418, PP242, BAY11-7082, rapamycin, and anti-actin were provided from Sigma Chemical Co. (St. Louis, MO, USA). Anti-PARP, anti-Bcl-xL, anti-DR5, anti-cIAP1, anti-caspase-8, anti-phospho-GSK3β, and anti-GSK3β were provided by Cell Signaling Technology (Beverly, MA, USA). Anti-Bim, anti-Bax, and anti-XIAP were obtained from BD Biosciences (San Jose, CA, USA). Anti-Mcl-1, anti-Bcl-2, anti-cIAP2, and anti-Cbl were purchased from Santa Cruz Biotechnology (St. Louis, MO, USA). SB203580, SP600125, and anti-c-FLIP(L) were obtained from Enzo Life Sciences (San Diego, CA, USA). Anti-DR4 were obtained from Abcam (Cambridge, MA, USA). pCMV-Myc-Cbl plasmid was a gift from Dr. S. J. Kim (CHA University, Korea). GSK3betaS9A (1016) was a gift from Scott Friedman (Addgene plasmid # 49492; http://n2t.net/addgene:49492; RRID: Addgene_49492) [36 (link)].

Dulbecco’s modified Eagle’s medium (DMEM; #11965-084) was purchased from Life Technologies^TM (USA). The α-Tubulin antibody (#T5168) and AR-A014418 (#487021-52-3, an inhibitor of GSK3) were purchased from Sigma-Aldrich (USA). Antibodies against GSK3β (#9315) and MMP-2 (#4022) were purchased from Cell Signaling Technology (USA). Antibodies against CXCR4 (#ab2074) was purchased from Abcam.

Neurite degeneration was defined by beading (swellings along neurite) and fragmentation as previously described [60 (link), 68 (link), 69 (link)]. Briefly, cultured neurons were incubated by ammonia with or without pre-incubation of the calpain inhibitor (ALLM, 10 μM, Sigma), the pan-caspase inhibitor (ZVAD, 20 μM, Sigma) and the GSK-3 inhibitor (AR-A014418, 10 μM, Sigma) for 30 min, then neuronal morphology was evaluated. For calcium release indication, calcein-AM and Fluo-4 (all purchased from Thermo Fisher Scientific, Carlsbad, CA, USA) was applied to cultured neurons. Briefly, neurons grown on coverslips were incubated with calcium indicator 2 μM calcein-AM for 30 min at 37°C and viewed through using a fluorescent microscopy.

Pregnant Sprague-Dawley (SD) rats were purchased from Koatech (Pyeongtaek, Korea). Hippocampal tissues were dissected from embryonic day 17 (E17) SD rat embryos and dissociated with 0.25% trypsin solution (Invitrogen, Carlsbad, CA, USA). The isolated cells were plated on poly-ʟ-lysine-coated plates (Sigma-Aldrich, St. Louis, MO, USA) at a density of 5 × 10⁴ cells/well. Primary hippocampal neurons were cultured in Neurobasal media (Invitrogen) supplemented with 2 mM L-glutamine (Sigma-Aldrich), B27 supplement (Invitrogen), and 1× antibiotic Pen/Strep (Invitrogen) and were incubated at 37 °C in Dulbecco's modified Eagle's medium (DMEM; Welgene, Gyeongsan, Korea) under 5% circulating CO₂ for 10 days. The culture medium was changed every 2-3 days. The primary hippocampal neuron culture protocol was approved by the Institutional Animal Care and Use Committee of the Lee Gil Ya Cancer and Diabetes Institute, Gachon University (LCDI-2018-0137).
HEK293 cells and SH-SY5Y cells were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone Laboratories Inc, Logan, UT, USA) in a humidified atmosphere of 5% CO₂ at 37 °C and passaged for 2-3 days. The AR-A014418 (Sigma-Aldrich, St. Louis, MO, USA) and LiCl (Sigma-Aldrich, St. Louis, MO, USA), GSK-3 inhibitors, were treated with SH-SY5Y cell, and protein samples were extracted 4 hours later.

The antibodies used for blast cell identification by Flow Cytometry (FACSCanto II, Becton Dickinson, Rutherford, NJ, USA) were: anti-CD45-VioBlue, anti-CD45-APC-Vio770, anti-CD34-PerCP and anti-CD117-APC all from Miltenyi Biotech (Bergsch gradbach, Germany). For Western blot analysis, anti-β-catenin, anti- Ser675-phospho-β-catenin, anti-Ser33/37/Thr41-phospho-β-catenin, anti-non-phopho-β-catenin, anti-GSK-3β, anti-Ser9-phospho-GSK-3β, anti-GSK-3α, anti-Ser21-phospho-GSK-3α anti-Histone H3 antibodies and Alexa 488-conjugated secondary antibodies were from Cell Signaling (Danvers, MA, USA); anti-GAPDH, and HRP-conjugated secondary antibodies against mouse or rabbit were from Sigma-Aldrich (Darmstadt, Germany). Wnt modulators used for proliferation and vitality assays, i.e., Wnt-3a, PNU-74654, Niclosamide, IWP-2, Lithium Chloride (LiCl), and AR-A014418, were all purchased from Sigma-Aldrich. For the analysis of cell death, Propidium iodide (PI) and FITC-conjugated Annexin V were from Miltenyi Biotechnology. CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay (MTS, Eden Prairie, MN, USA) was from Promega (Promega, Milano, Italy). Cytarabine (Ara-C) and Idarubicin (Ida) were provided by the Pharmacy Unit of the University Hospital of Verona.