Lenalidomide-induced up-regulation of CD154 is dependent on PI3-kinase. (A) CLL cells were treated with vehicle or 0.5μM lenalidomide for 8 hours. Cell lysates were subjected to immunoblot analysis. (B) IKK-β was immunoprecipitated from cell lysate derived from vehicle or 0.5μM lenalidomide-treated CLL samples and tested for the capability to phosphorylate the GST-IκBα substrate in a kinase buffer containing 20μM ATP and 10 μCi (0.37 MBq) of [γ32P]ATP. The left panel shows a representative experiment of the IKK kinase activity. The graph shows fold change in IKKβ activity in the lenalidomide-treated group compared with vehicle control (n = 8, P = .01). (C) IKK substrate IκB is degraded after lenalidomide treatment. The panel shows 3 representative CLL patients treated with 0.5μM lenalidomide or the vehicle control for 12 hours. (D) After 48 hours with lenalidomide 0.5μM and vehicle control with and without 25μM LY294002, transcription was inhibited by the addition of ActD and 10⁷ cells were collected for each time point over a 4.5-hour time course. The percentage of RNA remaining from each time point is plotted (n = 4, P = .045). (E) A total of 5 × 10⁴ lenalidomide-treated CLL B cells (L) or vehicle control CLL B cells (VC) were irradiated (20 Gy) and placed in culture with 5 × 10⁴ targets, purified B cells from the healthy donor, in the absence or presence of PWM and 25μM LY294002. IgM production was assayed by ELISA from supernatants obtained after 7 days of coculture (n = 8, P = .001). Error bars represent SD.