Evaluation of the status of the DNA in the endothelial cells after exposure to the E1⁻E4⁺ AdNull vector or no vector (“control”). (A) Effect of AdNull on DNA synthesis of subconfluent endothelial cells. Cells were exposed to the AdNull vector (50 pfu/cell) or no vector (“control”) and were cultured in 96 well plates in growth factor medium or growth factor–free medium. After 24 hours, [³H]thymidine was added, and the incubation was continued for 24 hours. The data represents the mean ± standard error of the mean of triplicate measurements. (B) Effect of AdNull infection on the status of cellular DNA. Endothelial cells cultured in 6 well plates were exposed to the AdNull vector as per panel A and were evaluated by flow cytometry after propidium iodide staining. Shown are data for the confluent cultures of endothelial cells infected with AdNull in growth factor medium or growth factor–free medium. The data for day 1 is displayed with a linear abscissa to best show the >2n peaks; the data for day 5 and day 12 is displayed with a log scale to best show the <2n fragmented DNA. The <2n, 2n, and 4n DNA peaks are indicated. (C) Evaluation of apoptosis in cells infected with E1⁻E4⁺AdNull vector. Subconfluent endothelial cells cultured in poly-D-lysine–coated glass coverslips were infected with E1⁻E4⁺ AdNull vector as described in the Fig 1 legend. After vector infection, cells were maintained in growth factor medium for 10 days or in growth factor–free medium for 1 day, and apoptotic cells were identified by assessment of free DNA 3′-OH ends.