Siβ catenin

pCI/β-catenin and its mutants tagged with HA at N-terminus were constructed by PCR amplification from pcDNA3/β-catenin and confirmed by DNA sequence analysis. pGL4-1.0, pRL-TK (thymidine kinase promoter driven Renilla reniformis luciferase) and dual luciferase assay kit were bought from Promega (Madison, WI, USA). Luciferase driven by different length of or mutated promoter of human PME-1 in pGL4.10 were constructed by using human blood genomic DNA, purified by Spin Column Blood Genomic DNA Purification Kit (Sangon Biotech, China) as template and confirmed by sequencing. Monoclonal anti-PME-1, polyclonal anti-GAPDH, monoclonal anti-demethylated-PP2Ac (DM-PP2Ac), siGSK-3β, siGSK-3α and siβ-catenin were bought from Santa Cruz (Santa Cruz, CA, USA). Polyclonal anti-β-catenin, anti-Non-pS-β-catenin (dephosphorylated at S33/S37/T41), anti-GSK-3β and anti-pS9-GSK-3β were purchased from Cell Signaling Technology (Danvers, MA, USA). Monoclonal anti-HA was from Sigma (St. Louis, MO, USA). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). ECL (enhanced chemiluminescence) kit was from Thermo Fisher Scientific (Rockford, IL, USA).

For transient RYK overexpression, cells were transfected with 4 μg of pcDNA3-h-RYK or pcDNA3. Ryk cDNA was kindly provided by Dr Stacker [28 (link)]. Lipopfectamin 2000 (Invitrogen, Thermo Fisher, Milan, Italy) was used for U87MG and patient-derived stem cells; X-tremeGENE9 DNA Transfection Reagent (Roche, Milan, Italy) was used for U251MG and AM38 cell lines.
To transiently knockdown RYK or β-catenin cDNA, 100nM of siRyk (Ambion, Thermo Fisher), siβ-catenin (Santa Cruz Biotechnology, MA, USA), or siRNA control (Ambion, Thermo Fisher) were transfected using Lipopfectamin 2000 for patient-derived stem cells or Oligofectamine (Invitrogen, Thermo Fisher) for all other cell lines.

The rabbit anti-human RNF146 polyclonal antibody was purchased from Abcam (Cambridge Science Park). Anti-Axin and anti-β-catenin antibodies were purchased from BD Transduction Laboratories (San Jose, CA, USA). Anti-CyclinA, anti-CyclinB, anti-CyclinD1, and anti-pRB antibodies were from Cell Signaling Technology, Inc. (Boston, MA, USA). Anti-CDK4, Anti-CDK6, Anti-TIMP-1, Anti-CyclinE, siAxin, siβ-catenin, and siTCF4 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against RhoA, RhoB, RhoC, and Rock1 were from Proteintech (Chicago, IL 60612, USA). Antibodies against CDK2, P21, matrix metalloproteinase 2 (MMP2), MMP7, and MMP9 were from NeoMarkers (Palo Alto, CA, USA). Anti-P27 and anti-P53 were from Thermo Fisher Scientific (Fremont, CA, USA). The recombinant overexpression plasmids pEX4-hRNF146 and pGPHI-shhRNF146 and control empty plasmids were constructed by GenePharma (Shanghai, China). The immunohistochemical secondary antibody kit was purchased from Dako (Copenhagen, Denmark).