Figure 7. HF induces the dephosphorylation of Bad and Akt, and inhibits Akt1 kinase activity.

(A) U937 cells were treated with 1, 1.4 or 2 µg/ml HF or 100 nM F for 24 h, after which lysates were subjected to Western blots using antibodies recognizing Ser¹³⁶-p-Bad, Bad, Ser⁴⁷³-p-Akt1, Akt1 and actin. One experiment representative of three is shown. (B) Akt1 kinase activity was determined using a kinase assay kit obtained from Enzolifesciences France using recombinant active Akt1 in the absence or presence of increasing concentrations (0.5–5.5 µM) of Akt1/2 inhibitor Akt-I-VIII, F (flavopiridol), HF or vehicles EtOH (for HF) and DMSO (for F and Akt-I-VIII). Of note, 2.5 µM of HF corresponds to 1.4 µg/ml. Percent of Akt kinase activity was determined. (C) The percentage of apoptotic cells was determined after annexin-V-FITC/PI staining and cytometry, under control conditions, or 1.4 µg/ml HF or Akt-I-VIII (2.5, 5 and 10 µM) and in combination treatment for 48 h. Data are the mean ± SD of results from 2 independent experiments.