NVP-BEZ235 decreases DNA synthesis and triggers cytotoxicity in WM cells and IgM-secreting cell lines, without affecting normal cells. (A) Thymidine uptake assay. BCWM.1 cells were cultured with NVP-BEZ235 (0.1-400nM) for 24 to 72 hours. (B) Thymidine uptake assay. IgM-secreting cell lines (MEC.1; RL) were cultured with NVP-BEZ235 (0.1-400nM) for 48 hours. (C) Cytotoxicity was assessed by MTT assay. BCWM.1 cells were cultured with BEZ235 (0.1-400nM) for 24 to 48 hours and 48 to 72 hours. (D) Cytotoxicity was assessed by MTT assay. IgM-secreting cell lines (MEC.1; RL) were cultured with NVP-BEZ235 (0.1-400nM) for 48 hours. (E) BCWM.1 cells were transfected with either scramble probe or mTOR siRNA. Untransfected BCWM.1 cells were used as control. Cells were treated with NVP-BEZ235 (2.5-100nM) for 48 hours, and cytotoxicity was assessed by MTT assay. Whole-cell lysates were subjected to Western blotting using anti-mTOR and α-tubulin antibodies (inset [E]). (F) BCWM.1 cells were transduced with Akt short hairpin RNA. Mock: control plasmid. BCWM.1-transfected cells or BCWM.1 control cells were treated with NVP-BEZ235 (2.5-100nM) for 48 hours, and cytotoxicity was assessed by MTT. Whole-cell lysates were subjected to Western blotting using anti-Akt and α-tubulin antibodies (inset [F]). (G) Freshly isolated BM CD19⁺ tumor cells from 4 patients with WM were cultured with NVP-BEZ235 (5-50nM) for 48 hours. Cytotoxicity was assessed by MTT assay. (H) Freshly isolated peripheral blood–derived CD19⁺ from 4 healthy donors was cultured with NVP-BEZ235 (2.5-1000nM) for 48 hours.