Activated Notch induces cell cycle reentry in quiescent cardiomyocytes. (A–D) NRVCs were either uninfected (A and E) or infected with Ad-βGal (B and F) or Ad-N2_ICD (C and G). 48 h after infection, cells were stained with mouse anti-MF20 (green, Alexa 488) and propidium iodide (PI; A–C). The number of MF20⁺ cells (green, Alexa 488) was >90% in all cases. DNA content was analyzed for the MF20⁺ population (insets) and the percentage in S/G₂/M phase for each condition was calculated (D). The examples shown are representative of more than five experiments with similar outcomes. (E–H) Examples of NRVCs as in A–D stained with MF20 (red, Alexa 594) and Ki67 (green, Alexa 488; E–G) 48 h after infection, and the percentage of Ki67⁺ cells within the MF20⁺ population was measured (H). The examples shown are representative of more than five experiments with similar outcomes. (I) 12 h after infection, NRVCs were transfected with 0.6 μg of the pHes1-Luc or pΔHes1-Luc plasmids and 0.2 μg pGL3-Renilla-Luc. Luciferase activities and protein expression of Notch2 and V5 epitope (inset) were determined 12 h later. Firefly activity was normalized using Renilla luciferase activity. (J) Examples of NRVCs as in A–D stained with V5 (red, Alexa 594), Notch2, (green, Alexa 488), and DAPI (blue) 48 h after infection. (K) Confocal images showing the persistence of Ki67⁺ (green, Alexa 488) and MF20⁺ (red, Alexa 594) cardiomyocytes in differentiating mESCs expressing N2_ICD from the MLC2V promoter compared with typically quiescent cardiomyocytes in cultures harboring only the REX promoter–Bla^r gene. Error bars indicate ±SD across experimental replicates. Bars: (E–G and K) 10 μm; and (J) 50 μm.