Induction of BIM is required for direct apoptosis induced by IL-21. (A) IL-21 induces BIM induction. CD19⁺ B-CLL cells were stimulated with media, IL-21 (100 ng/mL) for 24 hours. Cells were lysed in appropriate buffers and analyzed by Western blotting using specific mAb for Bim and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described in Methods. Figure shows data from a representative responding and nonresponding patient. Numbers below the lanes represent the fold increase in Bim levels in IL-21–stimulated CLL cells relative to unstimulated cells from the same patient and normalized to GAPDH levels. Data are expressed in relative densitometric units. (B) BIM siRNA protects CD19⁺ B-CLL cells from IL-21–mediated direct cytotoxicity. CD19⁺ B-CLL cells were mock transfected (no siRNA) or were transfected with nonsense siRNA or BIM siRNA (n = 3). The CLL cells transfected with Bim siRNA are resistant to the IL-21–mediated direct cytotoxicity compared with scrambled or mock-transfected cells. (C) Western blot analysis of BIM protein expression in nonsense siRNA, BIM-specific siRNA, and mock-transfected CLL cells. The CLL cells mock transfected or nonsense siRNA or BIM siRNA and treated with IL-21 or media therapy for 72 hours and analyzed by immunoblot using BIM specific antibody. GAPDH panel represents the loading control. (D) The bar graph representing the fold increase in the BIM with and without IL-21 is shown. Results shown are representative of 3 independent patients.