Anti human β catenin

DMEM culture medium and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY). The doxorubicin (DOX, catalog #ST1285), CCK8 (catalog #C0043), apoptosis detection kit (catalog #C1062L), TOPFlash (catalog #D2501) and FOPFlash (catalog #D2503) was purchased from Beyotime (Shanghai, China). Anti-human β-catenin (catalog #8480), cyclin D1 (catalog #55506), cyclin B1 (catalog # 12,231), survivin (catalog # 2808), c-Myc (catalog #18583), CDK2 (catalog #18048), p53 (catalog #2527), β-actin (catalog #4970) and Histone H3 (catalog #4499) antibodies were bought from Cell Signaling Technology (Beverly, MA, United States). FH535 (catalog #HY-15721) and BML-284 (catalog #HY-19987) was purchased from Med Chem Express (MCE, United States). Humantenidine, gelsemine, koumine, gelsenicin, gelsevirine, and sempervirine (HPLC≥98%) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. The roots and stems of G. elegans were bought from a commercial source and authenticated by the Department of Pharmacognosy, School of Pharmacy, Fujian Medical University as previous reported (Liu Hao et al., 2008 ). Other chemicals were of analytical grade and commercially available.

After undergoing the treatment described earlier, DPCs were washed with ice-cold 1× PBS and lysed with RIPA buffer (cat# 89901, Thermo Scientific, Waltham, MA, USA) containing a protease and phosphatase inhibitor cocktail (cat# 1861281, Thermo Scientific). Protein concentrations were determined using a BCA Protein assay (cat# 23225, Thermo Scientific). The lysates were separated using Novex, NuPAGE, and Bolt precast gels (Invitrogen, Carlsbad, CA, USA) under denaturing conditions and transferred to nitrocellulose membranes. After blocking with 5% bovine serum albumin solution for 1 h at room temperature, the membranes were immunoblotted with various antibodies (anti-human phospho-GSK-3β [SER9], cat# 9323; anti-human β-catenin, cat# 9562; anti-human phosphor-AKT [SER473], cat# 9271; anti-human cyclin D1, cat# 2978; and anti-human GAPDH; cat# 5174, Cell Signaling, Danvers, MA, USA) overnight at 4 °C, and then probed with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The bands were visualized using an enhanced chemiluminescence immunoblotting system (GE Healthcare Life Sciences, Buckinghamshire, UK).

Human Wnt3a recombinant protein (rWnt3a), secreted frizzled-related protein-3 (sFRP-3), and dickkopf-1 (Dkk-1) were purchased from R&D Systems (USA). The recombinant rhTGF-β3 protein was purchased from Peprotech (Rocky Hill, NJ, USA). Fetal bovine serum (FBS) and Eagle's minimal essential medium (MEM) were purchased from HyClone (South Logan, UT, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma-Aldrich (St. Louis, MO). MicroBeads conjugated to monoclonal mouse anti-human CD105 and CD166, anti-mouse secondary antibodies coupled to magnetic beads, and primary antibodies anti-human CD105-PE, anti-human CD166-FITC, anti-mouse CD105, anti-mouse CD166 were obtained from Miltenyi Biotec (Germany), and anti-human β-catenin was obtained from Cell Signaling Technology (USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG was provided by Santa Cruz (USA). Trizol and Superscript III kits were purchased from Invitrogen (USA). The RNeasy mini kit was obtained from Qiagen (Netherlands).