Morphology, PML-RAR levels, growth curves, and apoptosis of NB4 and NB4.437r cells in the absence and presence of CD437. (A) NB4 or NB4.437r cells (1 × 10⁵/mL) were treated for 6 hours with vehicle or with CD437 (10⁻⁶ mol/L) as indicated. Cells were stained with May–Grunwald–Giemsa and photographed (magnification, ×400). Arrowheads in microscopic fields corresponding to CD437-treated NB4 cultures indicate apoptotic cells or apoptotic bodies. (B) NB4 or NB4.437r cells were treated with CD437 (10⁻⁶ mol/L) for the indicated amounts of time. Cell extracts were subjected to Western blot analysis. The filter was sequentially challenged with an anti-RARα and an anti-actin antibody. The position of the intact PML-RARα protein and a specific degradation product are indicated with a dot and an asterisk, respectively, on the right. The positions of appropriate molecular weight markers are indicated on the left. (C) Growth curves of NB4 in control conditions (open circles) and NB4.437r cells in the absence (solid circles) or presence (solid triangles) of CD437 (10⁻⁶ mol/L). The results are the mean ± SD of 3 separate culture dishes. (D) Surface expression of phosphatidylserine in NB4 and NB4.437r cells after treatment with CD437. NB4 (circles and diamonds) or NB4.437r (triangles and squares) cells (5 × 10⁵/mL) were treated for the indicated amounts of time with dimethyl sulfoxide as vehicle (open symbols) or CD437 (10⁻⁶ mol/L) (solid symbols). The number of viable (diamonds and squares), apoptotic (triangles and circles), and necrotic cells (numbers in parentheses) was determined by flow cytometry after staining with fluoresceinated annexin-V and PI. Viable cells are PI⁻ and annexin-V⁻; apoptotic cells are PI⁻ and annexin-V⁺(PI⁻/annexin-V⁺); the necrotic cells are PI⁺ and annexin-V⁺. Flow cytometric analysis was performed as in the inset of Figure 1. Data are representative of at least 2 independent experiments with identical results.