(A) Promoter structure of the Hes5, 2xTP1(H-T), and 4xTP1(H-T) luciferase constructs. Red dots indicate nucleotide differences between the human and mouse Hes5 SPS sites. Suspected RBPJ binding sites are highlighted in yellow and arrows depict directionality of RBPJ binding sites. (B) HEK293T cells were transfected with either the mouse (mHes5(SPS)) or human (hHes5(SPS)) sequence-paired site constructs or with the 2xTP1(H-T) or 4xTP1(H-T) head-to-tail constructs and WT or dimer-incompetent versions of NICDs. Shown is the average +/- SE of n = 4 independent experiments. * <0.05, ** <0.01, *** <0.001, student’s t-test. (C) HEK293T cells were transfected with various NICDs and Hes5 SPS constructs with 11–36 bp spacer regions (5 bp increments), n = 6.