For overexpression of GPR50, the pGEM-T Easy vector (Promega) was used to clone the complete GPR50 coding sequence using the primers listed in Table S2 . Afterward, the complete coding sequence (without the termination codon) was further subcloned into the pCDH-EF1-MCS-T2A-copGFP lentiviral vector (System Biosciences) using XbaI and EcoRI restriction enzymes and the primers listed in Table S2 .
For overexpression of JAG1, DLL1, NICD, and ADAM17, cells were incubated overnight to a cell density of 2 × 105 cells per well in a 24-well culture plate. The cells were then transfected with the expression vectors for hemagglutinin (HA)-JAG1, GFP-DLL1, FLAG-NICD (kind gifts from Professor Hee-Sae Park, Chonnam National University, Republic of Korea), and FLAG-ADAM17 (plasmid number 31713; Addgene, Watertown, MA, USA)88 (link) using the HyliMax transfection reagent in a 1:3 ratio (Dojindo, Kumamoto, Japan), according to manufacturer’s instructions. After 48 h of transfection, the transfected cells were ready for use in further experiments.
For overexpression of JAG1, DLL1, NICD, and ADAM17, cells were incubated overnight to a cell density of 2 × 105 cells per well in a 24-well culture plate. The cells were then transfected with the expression vectors for hemagglutinin (HA)-JAG1, GFP-DLL1, FLAG-NICD (kind gifts from Professor Hee-Sae Park, Chonnam National University, Republic of Korea), and FLAG-ADAM17 (plasmid number 31713; Addgene, Watertown, MA, USA)88 (link) using the HyliMax transfection reagent in a 1:3 ratio (Dojindo, Kumamoto, Japan), according to manufacturer’s instructions. After 48 h of transfection, the transfected cells were ready for use in further experiments.