(A) Expression of IκBα in PAEC sensitizes them to TNF-mediated apoptosis. Noninfected or rAd.IκBα-infected PAEC were treated for 7 hours with TNF (100 U/mL). Cells were then obtained as described and assessed for apoptosis-induced DNA fragmentation. TNF treatment did not affect the percentage of cells in the A₀region, whereas this percentage was increased to 42% in IκBα-expressing PAEC. (B) Infection of cultured PAEC with rAd.A20 and rAd.IκBα results in the coexpression of both transgenes as assessed by Northern blot analysis. (C) Coexpression of A20 in rAd.IκBα-infected EC reverts their phenotype to resistance against TNF-mediated apoptosis. PAEC were cotransduced with the rAD.IκBα at MOI of 500 for each virus. Noninfected PAEC or PAEC infected with the rAd.IκBα (500 MOI) alone or in combination with the rAd.A20 (500 MOI) or rAd.β-gal (500 MOI) were treated with TNF for 7 hours, after which time cell viability was assessed by crystal violet uptake. Results show that coexpression of A20 in rAd.IκBα-infected PAEC results in a significant increase in cell viability following TNF treatment (60% ± 2) as opposed to cells cotransduced with the rAd.β-gal, where only 19% of the cells were still viable (a percentage that is not significantly different from the cells transduced with the sole rAd.IκBα [28% of viable cells]). Results are also expressed as percentage of survival compared with the noninfected, nontreated (control) PAEC whose values were considered to represent 100% of cell survival. Results shown are representative of three independent experiments.