Smac enhances grzB-induced caspase activity and cell death. (A) Cytosolic extracts of L428 HL cells were prepared, and the protein content was normalized (10 μg/μL). Caspase activation was initiated by addition of cytochrome c/dATP or recombinant grzB (20 ng) in the absence or presence of Smac N7 peptide (10 μM). After incubation for 1 hour at 30°C, relative caspase-3 activity was measured by using DEVD-AFC as substrate. (B) Cells (5 × 105) were transfected using Chariot as vehicle, with β-galactosidase alone, in combination with either recombinant grzB or Smac N7 peptide, or in combination with recombinant grzB and Smac N7 peptide. Cell death was determined after 2 hours. Data are mean ± SD values from 3 individual experiments performed in triplicate. (C) L428 HL cells were transiently transfected with Ub-Smac-dsRed or Ub-dsRed (red). Cells were left untreated or treated with grzB/Ad. After fixation, nuclei were stained (Hoechst 33258) (blue) and analyzed by a motorized inverted microscope (Olympus Ix81; Tokyo, Japan) using a 63×/1.40 numerical aperture Planapo oil objective. Images were acquired using analy-SIS software (Soft Imaging System, Münster, Germany) and were further processed and assembled using Power-Point (Microsoft, Redmond, WA).
