Sb415286

Proteasomal inhibitor MG132 and GSK3β inhibitor SB415286 were purchased from Selleckchem.com and TOCRIS bioscience (Bristol, UK), respectively. Lithium chloride was obtained from Calbiochem (La Jolla, CA). Cycloheximide was purchased from Sigma (St. Louis, MO). Human and mouse Wnt3a was purchased from R & D Systems (Minneapolis, MN). Antibodies were purchased as follows: anti-CyclinD1, anti-HA, anti-c-Myc, anti-RhoA, and anti-poly (ADP) ribose polymerase (PARP) from Santa Cruz Biotechnology (Santa Cruz, CA); anti-STRAP and anti-β-catenin from BD Transduction Labs (San Jose, CA); anti-phospho-β-catenin (Ser33/37/Thr41), anti-GSK-3β, anti-APC and anti-phospho-GSK-3β (S9) from Cell Signaling Technology (Danvers, MA); and anti-β-actin and anti-Flag antibodies from Sigma (St. Louis, MO).

Sec-O-glucosylhamaudol (purity ≥95%) was obtained from Nature Standard (Shanghai, China). Alpha modification of Eagle’s minimum essential medium (α-MEM), fetal bovine serum (FBS), and penicillin and streptomycin (PS) were purchased from Gibco (Grand Island, NY, USA). Recombinant murine M-CSF and RANKL were from Prospec (Rehovot, Israel). SB415286 was purchased from Selleckchem (Houston, TX, USA). LPS from Escherichia coli O55:B5, 2,5-diphenyltetrazolium bromide (MTT), zoledronic acid (ZOL), tartrate-resistant acid phosphatase (TRAP) staining kit, rhodamine phalloidin, 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI), and all other chemicals were obtained from Sigma-Aldrich (St Louis, MO, USA). Antibodies against phospho-P65, NFATc1, c-Fos, and PP2B-Aα were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Primary HLECs (CC-2527; Lonza, Allendale, NJ) were maintained in EBM-2 medium with a GM-2 Bullet Kit (Lonza; Walkersville, MD). All cultures were maintained in a humidified 5% CO₂ incubator at 37 °C and routinely passaged when 80–90% confluent. Stable shControl and shAkt1 (ACGCTTAACCTTTCCGCTG) HLEC cells were generated using SMART vector 2.0 lentivirus particles (10⁹ p.f.u.) (Thermo Scientific, Waltham, MA). Lentiviral particles were mixed in 1 ml Hyclone SFM4Transfx-293 (Fisher, Hanover Park, IL) and added along with 1 µl Polybrene (10 mg/ml, American Bioanalytical, Natick, MA). Three days later, transfection efficiency was tested through Turbo-GFP expression and subjected to 4 µg/ml puromycin (Life Technologies, Grand Island, NY) selection until all the cells expressed GFP. Pharmacological inhibition of Akt activity (phosphorylation) was achieved by treating the cells for 24 h with 10 µM MK-2206, an Akt inhibitor widely used in the clinical trials and GSK-3 inhibition was achieved by using 10 µM SB415286 (Selleckchem, Houston, TX).

Using the jetPRIME transfection reagent (Polyplus), SH-SY5Y cells were transfected with a Renilla luciferase reporter plasmid carrying 869 bp of the KCTD12 promoter (SwitchGear), a firefly luciferase reporter plasmid, and a plasmid encoding CREB1-GFP (CREB, cyclic AMP-responsive element binding protein; GFP, green fluorescent protein) or control plasmid (pEGFP-C1, Clontech). After serum starvation overnight, a specific drug or inhibitor (LiCl, myoinositol, 8-bromoadenosine cAMP (8brcAMP) all from Sigma; SB415286 from Selleckchem) was added for different periods of time. Cells were lysed in reporter lysis buffer (Promega) containing a protease inhibitor cocktail (complete, EDTA-free, Roche) 2 days after transfection. Cell lysates were prepared to measure the luminescence of Renilla luciferase and firefly luciferase using a GloMax microplate scintillation and luminescence counter (Promega). The Renilla luciferase-derived luminescence from the KCTD12 promoter or control (Prom vector) was normalized to the luminescence measured from firefly luciferase, which accounted for differences in the transfection efficiency.

Antibodies include: anti-GSK-3β (D5C5Z, #12456), anti-phospho-GSK-3β (Ser9,5B3) (#9323) anti-Ki-67 (8D5, #9449) anti-Cleaved Caspase-3 (Asp175) (#9661) from Cell Signaling Technology (CST); anti-phospho-serine/threonine (ab15556) and anti-phospho-serine (ab9332) from Abcam; anti-Flag^® M2 from Sigma-Aldrich; anti-RARB (A1603) from ABclonal; anti-PARP1 (66520-1-Ig) and anti-His (66005-1-Ig) from Proteintech; anti-rabbit and anti-mouse secondary antibodies conjugated to horseradish peroxidase, anti-rabbit secondary antibodies conjugated to Cy3 from Invitrogen; anti-RXRα (ΔN197, sc-774; D20, sc-553), anti-RARα (C20, sc-551), anti-GFP (B-2,sc-9996), anti-c-Myc (9E10, sc-40), anti-β-actin (H196, sc-7210) and anti-GAPDH (FL-335, sc-25778) from Santa Cruz Biotechnology. Chemicals and other regents are: lipofectamin 2000 from Invitrogen; enhanced chemiluminescence (ECL), protein A/G agarose from ThermoFisher Scientific; sorafenib, BIO, SB415286 and tideglusib from Selleck Chemicals; 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 4,6-Diamidino-2-phenylindole (DAPI), 9-cis-RA, isopropyl-1-thio-b-D-galactopyranoside (IPTG) and LiCl from Sigma-Aldrich; cocktail of proteinase and phosphatase inhibitors from Roche; Dual-Luciferase Assay System Kit from Promega.

MTI-31 was synthesized in Shanghai Institute of Materia Medica, Chinese Academy of Sciences as previously described for example #44 [26 ]. Rapamycin, AZD8055, Lapatinib and GDC-0941 were purchased from BiochemPartner (Shanghai). SB415286 (Selleck Chemicals), Etomoxir (MedChem Express) and Oil red O (Sigma-Aldrich) were purchased. All other chemicals were purchased from Sigma-Aldrich unless otherwise specified. Inhibitors were dissolved in DMSO as 20 mM stock solution and were diluted before assays. pGIPZ- and/or pTRIPZ (inducible with doxycycline)-based lentiviral shRNA for human Bim ShRNA#3 and ShRNA#6 (V3LHS_411602, V2LHS_238924), Raptor ShRNA#2 (V3LHS_329849), Rictor ShRNA#4 (V2THS_225915) and non-targeting (NT, RHS4346) were obtained from Open Biosystems/GE Dharmacon.