GSK3-β contributes to AEB071-induced effects in B-CLL. (A-B) CD19⁺ cells from CLL patients (N = 9) were treated with AEB071 (2 μM) and stimulated with immobilized anti-IgM for 30 minutes or 24 hours. PKC phosphorylation at Thr638/641 and GSK3-β phosphorylation at Ser9 was assessed by immunoblot. Results from 2 patients are shown and band quantification is represented as fold change from unstimulated control (mean ± SD). (C-D) CD19⁺ cells from CLL patients (N = 6) were pretreated with media or okadaic acid (5 nM) for 2 hours, followed by incubation for 3 hours with AEB071 (2 or 5 μM). GSK3-β phosphorylation at Ser9 and AKT phosphorylation at Ser473 was assessed by immunoblot. Results from 2 patients are shown and GSK3-β band quantification is represented as fold change from unstimulated control (mean ± SD). (E) CD19⁺ cells from CLL patients (N = 4) were pretreated with media or okadaic acid (5 nM) for 2 hours, followed by incubation with AEB071 for 48 hours. Cell viability was measured by annexin/PI staining and expressed as mean ± SD. (F) CD19⁺ cells from CLL patients (N = 3) were treated with AEB071 (2 μM) or FTY720 for 5 hours. Enzymatic activity of PP2A in the cell lysates was measured by a nonradioactive assay as described in methods and expressed as fold change from untreated control (mean ± SD).