Lenalidomide leads to activation of the PI3K pathway via a PI3K-δ-dependent mechanism. (A) CD19⁺ cells from CLL patients (N = 9) treated with or without 0.5μM lenalidomide were examined for PI3K activity with and without the addition of 1 or 10μM CAL-101 to the lysate. Results were calculated relative to micrograms of protein. (B) CD19⁺ cells from CLL patients (N = 6) were incubated with or without 0.5μM lenalidomide and/or CAL-101 for 48 hours. AKT phosphorylation at Ser473 was assessed by immunoblot. Results are shown from one of 6 experiments. (C) CD19⁺ cells from CLL patients (N = 4) were incubated with or without 0.5μM lenalidomide and/or CAL-101 for 48 hours. GSK3β phosphorylation at Ser9 was assessed by immunoblot. Results are shown from one of 4 experiments. (D) CD19⁺ cells from CLL patients (N = 3) were transfected with siRNA targeted to PI3K-δ, PI3K-γ, or a nonsense target. p110δ protein expression was assessed by immunoblot. Results are shown from one of 3 experiments. (E) CD19⁺ cells from CLL patients (N = 3) were transfected with siRNA targeted to PI3K-δ, PI3K-γ, or a nonsense target and then incubated with or without 0.5μM lenalidomide for 48 hours. AKT phosphorylation at Ser473 was assessed by immunoblot. Results are shown from one of 3 experiments. (B-E) Quantification was done using the Alpha Innotech FluorChemQ MultiImage III System.