(A) Overexpression of A20 rescues CHX-sensitized EC from TNF-mediated apoptosis. Noninfected, rAd.A20-, and rAd.β-gal–infected confluent monolayers of PAEC were treated 48 hours following infection with 100 U/mL of TNF in the presence or absence of 2 μg/mL of CHX. Seven hours following treatment, cell viability was assessed using a vital dye (crystal violet) uptake assay as described. Results are expressed as percentage of survival compared with NI, nontreated (control) PAEC whose values were considered to represent 100% of cell survival. Results shown are the mean ± SEM of triplicate wells and are representative of three independent experiments. A20 expression significantly protects PAEC from CHX/TNF-induced cytotoxicity. No viable cells were seen in NI or rAdβ-gal–infected PAEC treated with CHX/TNF, as opposed to more than 60% to 70% viability in rAd.A20-infected PAEC treated or not with CHX (2 μg/mL) 30 minutes before the addition of TNF (100 U/mL). (B) Overexpression of A20 prevents apoptotic fragmentation of cellular DNA in CHX- and TNF-treated PAEC. Noninfected PAEC or PAEC infected with either rAd.β-gal or rAd.A20 were treated with CHX (2 μg/mL) or TNF (100 U/mL) either alone or in combination for 7 to 8 hours. Cells were then obtained and assessed for apoptosis-induced DNA fragmentation as described in Materials and Methods. The region below the G₁/G₀ peak, designated A₀, represents cells undergoing apoptosis with fractional DNA content and is presented as a percentage of the total events collected. Results obtained correlated with the crystal violet uptake data, validating its use for further experiments.