DNA damage checkpoint activation implicated in G₂/M arrest. (A–E) NRVCs were either infected with Ad-N2_ICD or left uninfected. 24 h later, 10 mM caffeine (C, D, H, and I) or control (A, B, F, and G) media were added. Cells were then cultured for an additional 24 h, by which time they were at P5, and stained with MF20 (red, Alexa 594) and for phospho-His3 (Ser10; green, Alexa488; A–D). The incidence of phospho-His3⁺, MF20⁺cardiomyocytes is shown (E). (F–J) Alternatively, a TUNEL assay (green, fluorescein) was performed before immunostaining with MF20 (red, Alexa594). Note that caffeine permitted the induction of phospho-His3 but triggered TUNEL reactivity. (K) N2_ICD (anti-V5 epitope) and cyclin D1 expression, and the corresponding levels of phosphorylated Cdc2 are shown for NRVCs prepared as in A–E. Elevated phospho-Cdc2 was observed with activated N2_ICD and reduced by caffeine to basal levels, which together is indicative that N2_ICD treatment activates the DNA damage checkpoint. α-actinin and nonspecific proteins are shown as loading controls. Error bars indicate ±SD. Bars, 25 μm.