Effect of A20 overexpression on apoptotic fragmentation. Overexpression of A20 prevents apoptotic fragmentation of cellular DNA in CHX/TNF-treated PAECs. (A) Expression of A20 was evaluated by Western blot analysis in HCAECs before and 3 and 6 hours following treatment with TNF and compared with A20 expression in NI PAECs and rAdA20-infected PAECs at an MOI of 500. In this system, rAd-mediated expression of A20 reached 2.6- to 4.3-fold that of the physiologically induced levels. Data shown are representative of 3 experiments performed. (B) Noninfected PAECs and PAECs infected with rAdβ-gal or rAdA20 were treated with CHX (2 μg/mL) and TNF (100 U/mL), either alone or in combination, for 6 hours. Apoptosis was assessed by DNA content analysis by means of flow cytometry. The region below the G₁/G₀ peak, designated A^o, represents cells undergoing apoptosis with fractional DNA content (less than 2N) and is presented as a percentage of the total events collected. A20 significantly (P < .0001) protected PAECs from CHX/TNF-mediated apoptosis. Expression of the A20 transgene in each experiment was assessed by Western blot analysis of cell extracts from rAdA20-infected PAECs at an MOI of 100 (arrows). Data shown are representative of 7 independent experiments.