Notch induces expression and nuclear localization of cyclin D1 and phosphorylation of Rb before cell cycle reentry. (A) The DNA content of the cardiac cells analyzed by flow cytometry (as in Fig. 1) at the indicated times after infection. One of two experiments with similar outcomes is shown. (B) Cyclin D1 and V5 epitope (N2_ICD) expression in NRVCs infected with Ad-N2_ICD at the indicated times after infection. (C) Time course of cyclin E expression after Ad-N2_ICD infection of NRVCs. (D) Examples of nuclear localization of cyclin D1 (arrows) in NRVCs infected with Ad-N2_ICD as compared with Ad-βGal–infected control cultures 48 h after infection. Immunostaining is shown for cyclin D1 (green, Alexa 488), MF20 (red, Alexa 594), V5 epitope tag on N2_ICD (far red, Cy5, shown as green), and DAPI (blue). Confocal microscopic images (insets) confirmed nuclear localization. (E) Phosphorylation status of Rb in NRVCs 30 h after infection with Ad-N2_ICD or 24 h after treatment with 10% FCS or 20 μM PHE. 5 mM EDTA in the lysis buffer prevents phosphorylation from occurring in the lysates. N2_ICD (V5 epitope), cyclin D1, phospho-Rb (Ser807/811, Ser795, and Ser780), Cdk4, Cdk6, and phospho-Cdc6 expression are shown. (F) Incidence of phospho-Rb (Ser807/811) in NRVC cardiomyocytes (MF20⁺ cells). Note that only N2_ICD induced significant levels of phospho-Rb. Error bars indicate ±SD. (G) Immunostaining with MF20 (red, Alexa 594) and for phospho-Rb (Ser807/811; green, Alexa 488) or cyclin D1 (green, Alexa 488). Note that cyclin D1 and phospho-Rb are present in the nuclei of N2_ICD-expressing cells (arrow) but not in the nuclei of cells cultured with high serum or PHE. Higher-magnification images are shown in insets with DAPI staining (blue). Bars: (D) 10 μm; (G, top) 50 μm; (G, bottom) 10 μm.