Lentiviral pLKO.1-puromycin negative control and human CDH2, CTNND1, CTNNB1, and CTNNA1 shRNA vectors were from Sigma-Aldrich (Table 2 ).
pLenti.CAG.H2B.Dendra2.W was from Addgene (RRID:Addgene_51005). N-cad WT or W161A mutated DNA was amplified by PCR from pCAG-Ncad WT or W161A-HA (Kon et al., 2019 (link)) and cloned into lentiviral pLenti-CAG-mCherry vector using NEBuilder HiFi DNA assembly (E2621; New England Biolabs) to generate pLenti-Ncad WT-HA-mCherry or pLenti-Ncad W161A-HA-mCherry.
To harvest lentiviral particles, lentiviral vector DNA was transfected with psPAX2 (RRID:Addgene_12260) and pMD2.G (RRID:Addgene_12259) packaging plasmids into HEK-293FT cells using Lipofectamine 2000 transfection reagent (11668019; Invitrogen) and D10 media lacking antibiotics. After 24 h, media were changed to glioma growth medium and the virus collected for a further 40 h. Culture media were collected and filtered through a 0.45-μM syringe filter. Glioma cells in a six-well plate were incubated with 500 μl of viral supernatant and 500 μl growth media for at least 48 h before selection. pLKO.1-shRNA-puromycin transduced cells were selected with 0.5 μM puromycin for 48 h. Cells expressing H2B-Dendra2, Ncad WT-HA-mCherry, or Ncad W161A-HA-mCherry constructs were sorted on a SONY MA900 Multi-Application Cell Sorter.
pLenti.CAG.H2B.Dendra2.W was from Addgene (RRID:Addgene_51005). N-cad WT or W161A mutated DNA was amplified by PCR from pCAG-Ncad WT or W161A-HA (Kon et al., 2019 (link)) and cloned into lentiviral pLenti-CAG-mCherry vector using NEBuilder HiFi DNA assembly (E2621; New England Biolabs) to generate pLenti-Ncad WT-HA-mCherry or pLenti-Ncad W161A-HA-mCherry.
To harvest lentiviral particles, lentiviral vector DNA was transfected with psPAX2 (RRID:Addgene_12260) and pMD2.G (RRID:Addgene_12259) packaging plasmids into HEK-293FT cells using Lipofectamine 2000 transfection reagent (11668019; Invitrogen) and D10 media lacking antibiotics. After 24 h, media were changed to glioma growth medium and the virus collected for a further 40 h. Culture media were collected and filtered through a 0.45-μM syringe filter. Glioma cells in a six-well plate were incubated with 500 μl of viral supernatant and 500 μl growth media for at least 48 h before selection. pLKO.1-shRNA-puromycin transduced cells were selected with 0.5 μM puromycin for 48 h. Cells expressing H2B-Dendra2, Ncad WT-HA-mCherry, or Ncad W161A-HA-mCherry constructs were sorted on a SONY MA900 Multi-Application Cell Sorter.
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