Selective activation of initiator caspases in Lptn-induced apoptosis.

(A) The Lptn-induced increase in DNA fragmentation is blocked by z-VAD-fmk; 25 μM of z-VAD-fmk (▪) or control (■) DMSO was preincubated with the cells prior to CD3 stimulation for 2 days, in the presence or absence of Lptn, followed by TUNEL staining. The panel is representative of 2 independent experiments. (B) Respective strong and weak processings of procaspases-9 and -8 in Lptn-mediated apoptosis. CD4⁺ T-cell lysates were made after the indicated times and conditions of activation and analyzed by Western blots that were sequentially probed for detection of expression of caspases-9 and -8, and the invariant PI3K control, using the antibodies described in “Materials and methods.” Arrows indicate the procaspases. No or little increased expression of procaspase-9, and decreased expression (2- to 3-fold) of procaspase-8 are observed on CD3 and CD28 costimulation in the presence of Lptn. This typical experiment is a representative of 3. (C) Kinetics of down-regulation of procaspase-9 expression by Lptn. The graph represents the relative levels of the caspase-9/PI3K signal ratio induced by the indicated stimuli, with error bars corresponding to SD. The results are representative of 3, 4, and 3 independent experiments at 0.5, 2, and 3 days, respectively. (D) An increase in caspase-9 activity is induced by Lptn. Cell lysates were prepared after the indicated times and conditions of activation, and analyzed in a caspase-9 specific colorimetric assay, as described in “Materials and methods.” For each condition, the graph represents the mean values from 2 independent experiments.