Degradation of PML–RAR- by CD437 in PR9 and NB4 cells. (A) NB4 cells (300,000/mL) were treated for the indicated amount of time with vehicle (DMSO, Control) or with 10⁻⁶ mol/L CD437. (B) NB4 cells (300,000/mL) were treated for 6 hours with vehicle (DMSO, Control) or with 10⁻⁶ mol/L CD437 following pretreatment for 2 hours in the absence or presence of z-VAD (100 μmol/L). (C) U937, PR9, and MT8 cells (300,000/mL) were treated for 16 hours with vehicle (DMSO, Control) or with 10⁻⁶ mol/L CD437 in the presence or absence of zinc sulfate (Zn, 100 μmol/L) as indicated. The percentage of cells showing nuclear fragmentation upon staining with DAPI was scored on parallel cultures. Each value represents the mean ± SD of three replicate cultures. ND, not determined. For Western blot analysis, cells were obtained, homogenized, and aliquots of the homogenate containing an equivalent amount of protein were subjected to polyacrylamide gel electrophoresis, electrotransfer to nitrocellulose membranes, and incubation with antibodies recognizing human RAR- and human actin (A). Alternatively, blots were sequentially probed with the two antibodies (B and C). The molecular weight of protein standards is indicated on the left. The results were replicated in a separate experiment.