Fig 5.

NOTUM is a direct target of RUNX2 and activates the expression of odontoblast marker Dspp in vitro. (A) Genome browser snapshots representing the peak of ATAC‐seq from PN7.5 control mouse molars colocalized with the RUNX2 binding site at the promoter region of NOTUM. (B) ChIP analysis revealed the binding of endogenous RUNX2 to the genomic loci of NOTUM. DNA before immunoprecipitation (input) and after immunoprecipitation with an anti‐RUNX2 or rabbit IgG was amplified by qPCR using primers that amplify the regions containing RUNX2‐binding motifs in the NOTUM promoter. The value of input was defined as 1, and relative levels are shown. (C–J) RNAscope in situ hybridization (red) and qPCR of Dspp in cultured dental pulp cells treated with CM, OM, and OM + NOTUM protein (C–F), as well as OM + control siRNA, OM + Runx2 siRNA, and OM + Runx2 siRNA + NOTUM protein (G–J), insets in C–E and G–I are enlarged images of the cells pointed to by arrows in the same image. Scale bars = 25 μm. CM = control growth media; OM = odontoblastic media.