Effect of CD437 on the levels and state of activation of caspase isoenzymes in NB4 and NB4.437r cells. NB4 (A) and NB4 or NB4.437r (C) cells (5 × 105/mL) were treated with vehicle (control), CD437 (10−6 mol/L), the caspase inhibitor z-VAD (100 μmol/L), or CD437 + z-VAD for the indicated amounts of time. The levels of the indicated caspase proenzymes and polyADP ribose polymerase (PARP) as well as relative degradation products were analyzed by Western blot analysis using specific polyclonal antibodies. Western blot filters were subsequently challenged with an anti-actin antibody to confirm equal protein loading in each lane of the gel. (B) The state of caspase activation was measured in NB4 cell extracts with fluorogenic peptide substrates specific for caspase-3 and caspase-7 (DEVD-amc), caspase-6 (VEID-amc), and caspase-8 (IETD-amc) after treatment with vehicle (open symbols) and CD437 (closed symbols) for the indicated amounts of time. Results are the mean ± SD of 3 separate culture dishes. Data are representative of at least 2 independent experiments with similar results.
