Recombinant human jag1

Human ESCC cells (KYSE510, KYSE410, KYSE450, and KYSE70) were obtained from the ATCC (Manassas, VA, USA) and the ECACC (Porton Down, Salisbury, UK) with proper authentication. These ESCC cell lines were selected for experiments based on their expression of NOTCH components and PAX9 (supplementary material, Figure S1). KYSE450 cells are known to carry a frame-shift NOTCH1 mutation (https://portals.broadinstitute.org/ccle). KYSE510 and KYSE410 were exposed to ethanol, dibenzazepine (DBZ; a chemical NOTCH inhibitor) (Cayman, Ann Arbor, MI, USA), or recombinant human JAG1 (a NOTCH ligand; R&D System, Minneapolis, MN, USA). The human JAG1 recombinant protein containing the signal peptide and extracellular domain of JAG1 fused at the C-terminus to the Fc portion of human IgG (R&D System) was immobilized to the plastic surface of the culture plates, by incubating plates with a solution of JAG1 (5 μg/ml) for 2 h at 37 °C. Cells were then seeded on JAG1-coated plates for 72 h. 3XFlagNICD1 overexpression plasmid (Plasmid #20183, Addgene, Watertown, MA, USA) [18 (link)], or RBPJ siRNAs (Thermo Fisher, Waltham, MA) were transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. All cell culture experiments were triplicated.