RBP-Jκ is insufficient to promote nuclear localization of cyclin D1. (A and B) Examples of NRVCs were transfected with 0.8 μg of plasmids encoding a myc epitope-tagged, constitutively active version of RBP-Jκ in pCDNA3 (pRBP-Jκ-VP16; A) or infected with Ad-N2_ICD (B) and stained 48 h later for cyclin D1 (green, Alexa 488), c-myc epitope (red, Alexa 594), MF20 (red, Alexa 594), and DAPI (blue). Note the absence of nuclear localized cyclin D1 in c-myc⁺ (RBP-Jκ-VP16⁺) cells. Circles indicate nuclear regions and arrows indicate nuclear localized cyclin D1. (C) Percentage of cardiomyocytes treated as in A and B showing nuclear versus cytosolic localization of cyclin D1. Error bars indicate ±SD. (D) NRVCs transfected to express a control DNA-binding mutant of RBP-Jκ (pRBP-Jκ-DBM) and stained as in A. Only a basal level of cyclin D1 was detected. Circles indicate nuclear regions. (E) Percentage of c-myc⁺/Ki67⁺ (transfected) and c-myc⁻/Ki67⁺ (untransfected) cells for pRBP-Jκ-VP16 (n = 69 and 107, respectively) and pRBP-Jκ-DBM (n = 57 and 106, respectively) as compared with Ad-N2_ICD infected (n = 100); results are representative of four trials. (F) RBP-Jκ and N2_ICD (0.5 μg of plasmid per transfection), as in A–E, showed expected activities on the pHes1-luciferase reporter (0.25 μg of plasmid per transfection); results are representative of two trials. Bars, 5 μm.