Ad-N2_ICD–infected NRVCs arrest at the G₂/M interphase. (A and B) 3 d after birth, NRVCs were infected with Ad-N2_ICD or Ad-β-gal, or not infected, and, 12 h later, treated with aphidicolin or nocodazole for an additional 36, 48, or 60 h. For nocodazole+ release, nocodazole was removed during the last 12 h of culture (see schematic in A, top). The percentages of cells in S/G₂/M phase in MF20⁺ (cardiomyocytes; A) and MF20⁻ (noncardiomyocytes; B) populations were determined from flow cytometry. Nocodazole did not increase the incidence of G₂/M cells among the N2_ICD-treated cardiomyocyte population, which is indicative of a block at the onset of M phase. The example shown is representative of four experiments with similar outcomes. (C–I) NRVCs were either left uninfected (C and F) or infected with Ad-βGal (D and G) or Ad-N2_ICD (E and H), cultured for 48 h, then stained with MF20 (red, Alexa 594) and for AuroraB (green, Alexa 488; C–E) or for phospho-His3 (Ser10; green, Alexa 488; F–H). The percentages of positive cells within the MF20⁺ population was then determined (I). Nuclear-localized AuroraB but not phospho-His3 was detected in response to N2_ICD. Error bars indicate ±SD. Bar, 25 μm.