Doxorubicin- or OKT3-induced apoptosis and CD95L expression.

(A) Doxorubicin- or OKT3-induced apoptosis. (upper panel) CD3+ (▪) or CD3- (□) H9 cells were incubated for the time points indicated on 96-well plates precoated with 100 μg/mL OKT3. (lower panel) CD3+ H9 cells were treated with doxorubicin (▪, 0.003 μg/mL; □, 0.01 μg/mL; x, 0.03 μg/mL; ○, 0.1 μg/mL; ■, 0.3 μg/mL). Apoptosis was determined by fluorescence-activated cell-sorting analysis of propidium iodide-stained DNA content. Percentage of specific apoptosis was calculated as follows: 100 × (experimental apoptosis [%] − spontaneous apoptosis [%]/100% − spontaneous apoptosis [%]). Data are the mean of triplicates; similar results were obtained in 3 separate experiments. (B) Doxorubicin- or OKT3-induced CD95 ligand expression. CD3+ H9 cells were treated with 0.1 μg/mL doxorubicin for the time points indicated. CD3+ or CD3− H9 cells were incubated for 6 hours in 75-cm² flasks precoated with 100 μg/mL OKT3. CD95-L mRNA expression was determined by reverse transcription–polymerase chain reaction. Expression of β-actin was used to control RNA integrity and equal gel loading. For Western blot analysis, 40 μg cell lysate protein per lane was separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). CD95-L protein was detected as 40 kD band by mouse anti-CD95-L monoclonal antibody (clone G247-4) and ECL. Expression of tubulin was used to control equal protein loading. (C) Dose response of doxorubicin-induced CD95 ligand expression. CD3+ H9 cells were treated with 0.003-0.1 μg/mL doxorubicin for 6 hours. 40 μg protein of cell lysates per lane were separated by 12% SDS-PAGE. CD95-L protein was detected as 40 kD band by mouse anti-CD95-L monoclonal antibody (clone G247-4) and ECL.