SMI-4a treatment induces apoptosis in pre–T-LBL. (A) May-Giemsa staining of 6812/2 and Jurkat was carried out after incubation with DMSO or 10μM SMI-4a in serum-free medium for 24 hours. Arrows indicate cells with nuclear condensation. (B) Jurkat cells were stained with annexin V and PI after treatment with DMSO or SMI-4a as in panel A. The R2 quadrant contains the cells that have undergone apoptosis. (C) PARP cleavage was examined in 6812/2 and Jurkat after incubation with DMSO or 10μM SMI-4a in the absence of serum for the indicated times. Western blots of extracts of these cells were probed with antibodies to PARP and GAPDH as a control. denotes holo- and cleaved PARP. (D) Jurkat cells were incubated with SMI-4a for 8 hours at the indicated concentrations. The caspase inhibitor zVAD-FMK (40μM) was added along with SMI-4a (0, 30μM) for the same time period. Extracts of these cells were Western blotted with antibodies to PARP and caspases-3 and -9. denotes the holo- and cleaved enzymes. GAPDH levels were measured as a loading control. (E) 6812/2 cells were incubated with 10μM SMI-4a in serum-free medium for 4 hours. Cells were lysed and the cytoplasmic proteins were extracted and separated from nuclear protein. Bax protein levels were detected by Western blotting. (F) Jurkat cells were transfected with either Bcl-2 or Bcl-xL expression vectors and permanent transfectants established. These cells were incubated with SMI-4a (30μM) in the absence of serum for 24 hours. Viable cells were identified by trypan blue exclusion. The experiment was repeated in triplicate and the standard deviation of the mean of these determinations is shown.