A20 protection from Fas- and FADD-induced cell death and caspase activation. A20 expression protects ECs from Fas- and FADD-induced cell death and caspase activation. BAECs were transfected with a control, a pcDNA3 plasmid or an A20 expression plasmid, and a β-gal expression plasmid. (A) Cotransfection with Fas alone (nontreated [NT]) resulted in cell death, which was enhanced by Fas cross-linking (αFas). Cell death was quantified by blue cell count per HPF. A20 significantly protected from Fas-mediated cell death. Data are expressed as mean ± SD of 3 HPFs per triplicate well. Results shown are representative of 3 independent experiments. (B) Similar results were obtained when cells were cotransfected with FADD. Cotransfection of BAECs with a dominant negative FADD was used as control. Caspase 8 (C) and caspase 3 (D) activities were determined in lysates of BAECs cotransfected with pcDNA3 or A20 plasmids and FADD expression plasmid by means of colorimetric assays based on the caspase-specific cleavage of labeled substrates (IETD-pNA for caspase 8 and DEVD-pNA for caspase 3). Expression of A20 significantly inhibited FADD-mediated activation of caspases 8 and 3. Data are expressed as mean ± SD of triplicate values. Results shown are representative of 4 independent experiments.