Induction of BIM expression contributes to TCR-induced apoptosis. (A) Mircoarray analysis of designated Bcl-2 family members was performed using RNA purified from activated human PBL either untreated (0 h) or stimulated with OKT3 for 6 h. Relative expression values normalized to reference RNA from normal human PBL are shown at left, fold change following TCR restimulation is quantitated at right. (B) Activated human PBL were stimulated with OKT3 for the indicated times, and whole cell lysates were prepared and immunoblotted for the proteins indicated on the right. All three isoforms of BIM (extra-long (EL), long (L), short (S)) were detected. Spot densitometry analysis of the ratio of BIM-EL to BCL-xL (normalized to β-actin loading control) is plotted below. (C) Activated human PBL were transfected with nonspecific (NS) or Bim-specific siRNA, rested 4 days, and then restimulated for 24 h with increasing doses of OKT3 in the presence or absence of SM1/23. Percent cell loss was calculated in triplicate by PI exclusion. Differences in apoptosis sensitivity (relative to NS alone) were statistically significant for each dose of OKT3 (p < 0.04), except for SM1/23 treated NS cells at 1 μg/ml. (D) Lysates from cells transfected in (C) were immunoblotted for BIM as in (B). β-actin serves as a loading control. (E) Average extent of TCR-induced apoptosis inhibition (relative to NS siRNA alone) is shown for each condition described in (D) for 6 different normal donor PBL tested.