Wnt3a 5036 wn

Methylthiazoletetrazolium (MTT), lithium chloride (LiCl), cytochalasin B (CytoB), L-glucose (L-Glu), glutamine (Glt), D-glucose and mannitol (Mntl) were purchased from Sigma-Aldrich (Sigma Aldrich, MO, USA). 6-Bromoindirubin-3′-oxime (BIO) was purchased from Merck Millipore (Merck Millipore, MA, USA). Antibodies against DKK4 (sc-25519), GLUT-1 (sc-7903), GLUT-2 (sc-9117), Cyclin D1(sc-717), CDK4 (sc-260), CDK6 (sc-7181), c-Myc (sc-764), Wnt3a (sc-28824), pJNK (sc-6254), JNK (sc-474), pMEK (sc-7995), MEK (sc-13069), Raf (sc-227), pERK (sc-7383), ERK (sc-154), pAkt (sc-7985), AKT (sc-8312), GSK3β (sc-9166), β-tubulin (sc-9104) and Histone H1 (sc-10806), HRP-conjugated (sc-2004), (sc-2031), (sc-2033), FITC conjugated (sc-3839) and Rhodamine conjugated (sc-358922) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, CA, USA). Antibody for β-catenin (610154) was purchased from BD Bioscience (BD Bioscience, NA, USA). Antibody for pGSK3β (9336S) was purchased from Cell Signaling technologies (Cell Signaling technologies, MA, USA). Wnt3a (5036-WN) and DKK4 (1269-DK) recombinant proteins were purchased from R&D Systems (R&D Systems, MN, USA). For developing stable cell lines, shRNA for DKK4 and control plasmids were purchased form Open Biosystems (Thermo Fisher Scientific, PA, USA).

The following antibodies were purchased from Cell Signaling Technology (Danvers, MA): normal rabbit IgG (# 2729) (for immunoprecipitation and ChIP), β-catenin (#8480) (for immunoblotting and immunoprecipitation), FTO (#31687) (for immunoblotting and RIP), EZH2 (#5246) (for immunoprecipitation and ChIP), TCF4 (#2569) (for ChIP), H3K27me3 (#9733) (for ChIP), H3K9me2 (#4658) (for ChIP), HK2 (#2867) (for immunoblotting and IHC), and Ki-67 (#9449) (for IHC). A mouse monoclonal antibody against tubulin (T9026) (for immunoblotting) was purchased from Sigma-Aldrich (St. Louis, MO). Rabbit antibodies recognizing c-Myc (ab32072) (for immunoblotting and IHC), Wnt-3a (ab219412) (for IHC) and FTO (ab124892) (for IHC) were purchased from Abcam (Cambridge, MA). Rabbit antibody recognizing YTHDF1 (17479-1-AP) (for RIP and immunoblotting) was purchased from Proteintech (IL, USA). RIPA lysis and extraction buffer (89901) and Pierce IP lysis buffer (87787) were purchased from Thermo Fisher Scientific (Waltham, MA). Protein A/G plus-agarose (sc-2003) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Wnt-3a (5036-WN) was obtained from R&D Systems (Minneapolis, MN). Cycloheximide (CHX) (HY-12320) and nitro blue tetrazolium chloride (HY-15925) were purchased from MedChem Express (Monmouth Junction, NJ). Agar (1182GR500) was purchased from BIO FROXX (Guangzhou, China).

Cell lines and patient derived samples were treated with the following prior to downstream assays: 50 ng/ml DKK1 (GF170, Milipore), 50 µg/ml Vantictumab (Oncomed), 100 nm Wnt3A (5036-WN, R&D systems), 100 µM LGK974 (S7143, Selleckchem), 10 ng/ml rIL15 (247-ILB, R&D systems), 10 ng/ml rIL1β (201-LB, R&D systems), 5 µg/ml IL1β neutralising antibody (MAB201, R&D systems), 5 µg/ml IL15 neutralising antibody (MAB-274, R&D systems), 5 µg/ml IL6 neutralising antibody (MAB2061, R&D systems), 5 µg/ml IL8 neutralising antibody (MAB208, R&D systems), 10 µg/ml Anakinra (Amgen, Cambridge, UK), 5 mM Sulfasalazine (Sigma), 10uM KG-501 (Sigma). Treatment times for individual assays are detailed below.