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.
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.