Pgl3.0 luciferase reporter vector

For the luciferase assay, the DNA fragments containing the R1–R10 regions of the NPPC promoter (according to the results of the ChIP assay) were amplified by PCR from mouse genomic DNA using specific primers and inserted into the pGL3.0 luciferase reporter vector (E1910; Promega, Madison, WI, USA). The forward primer contained a restriction enzyme site of MluI, and the reverse primer contained a restriction enzyme site of XhoI. The cDNA-derived mouse Smad3 sequence was amplified using PCR with the primer containing the MluI–KpnI restriction sites and inserted into the pcDNA3.1 vector (1332; Addgene, Cambridge, MA, USA). Primer sequences were listed in Supplementary Table S3. KK1 cells were maintained in cell culture medium at 37 °C with 5% CO₂. This cells were transiently transfected with Smad3 expression vector, Nppc luciferase reporter vector, and pTK-Renilla vector using Lipofectamine 2000 reagent (Thermo Fisher Scientific). An empty luciferase reporter vector was used as a control. After 24 h of transfection, the cells were harvested, and luciferase activities were measured using the Dual-Luc Assay Kit (E1960; Promega). Values shown by the fluc to rluc ratio were normalized to an empty luciferase reporter control.