Inhibition of PI3 kinase by LY294002 or Syk with piceatannol promotes synergistic apoptosis with Hu1D10 treatment and abrogates Hu1D10-induced phosphorylation of AKT. (A) CLL cells treated with (i) LY294002 25 μM, (ii) Hu1D10 plus antihuman Fc antibody (both 10 μg/mL), or (iii) Hu1D10 plus antihuman Fc antibody (both 10 μg/mL) plus LY294002 25 μM for 4 hours and were assessed for apoptosis using annexin V/PI staining followed by FACS analysis. Error bars indicate the SD among 4 different CLL samples. (B) CLL cells were treated for the indicated time with Hu1D10 plus antihuman Fc antibody (both 10 μg/mL). + indicates samples that were pretreated with LY294002 25 μM for 20 minutes. Cells were immediately lysed following antibody treatment. Samples were immunoblotted using standard techniques. Blots were probed with anti–phospho-AKT (Ser473), stripped, and then reprobed with anti-AKT. (C) Samples were incubated with the Syk inhibitor piceatannol (25 μg/mL) for 20 minutes prior to Hu1D10 plus antihuman Fc antibody (both 10 μg/mL) treatment. After 4 hours of antibody treatment, samples were stained with annexin V–FITC/PI and analyzed by FACS. Values indicate the percent apoptosis above the untreated sample. Error bars indicate the SD among 4 different CLL samples. (D) CLL cells were treated for the indicated time with Hu1D10 plus antihuman Fc antibody (both 10 μg/mL). If noted, samples were pretreated with piceatannol 25 μg/mL for 20 minutes. Blots were probed for phospho-AKT and AKT as noted.