Effect of CD437 on the phosphorylated form of Akt protein in NB4 and NB4.437r cells. Consequences of the inhibition of Akt phosphorylation by wortmannin on CD437-induced apoptosis. (A) NB4 cells (5 × 10⁵/mL) were treated with vehicle, CD437 (10⁻⁶ mol/L), the caspase inhibitor z-VAD (100 μmol/L), or the indicated combinations of the compounds for 1 hour or 4 hours. (B and C) NB4 or NB4.437r cells (5 × 10⁵/mL) were preincubated with vehicle or the PI3K inhibitor wortmannin (Wort, in B) for 1 hour. Subsequently cells were treated with vehicle, CD437 (10⁻⁶ mol/L), or the indicated combinations of the compounds for 1 hour. In A and B, the levels of the phosphorylated form of Akt (Akt_p) protein or of total Akt protein were determined by Western blot analysis using aliquots of the same cellular extracts and specific antibodies. Western blot filters were subsequently challenged with an anti-actin antibody to confirm equal protein loading in each lane of the gel. In C, the proportion of apoptotic cells was determined after staining of nuclei with DAPI. Results are the mean ± SD of 3 separate culture dishes. a, significantly higher than the relative control cultures (CD437⁻, Wort⁻) according to the Student t test (P < .01); b, significantly higher than the relative CD437-treated cultures (CD437⁺, Wort⁻) according to the Student t test (P < .01). Data are representative of 2 independent experiments with similar results.