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. Author manuscript; available in PMC: 2015 Mar 15.
Published in final edited form as: Clin Cancer Res. 2014 Mar 15;20(6):1576–1589. doi: 10.1158/1078-0432.CCR-13-0987

Fig. 5. BH3-only proteins Bim and Noxa are required for apoptosis induction by MLN4924.

Fig. 5

(A) CLL cells were transfected with two individual siRNA's against Bim or control siRNA using Amaxa program X-05. Immediately after nucleofection, cells were cultured on CD40L-expressing stroma for 24 hours, followed by incubation with 0.5 μM MLN4924 or vehicle control for 24 h. Whole-cell lysates were subjected to immunoblotting. A representative blot of three independent experiments is shown. (B-C) CLL cells were transfected with siRNA against Bim (N=7), Noxa (N=7), Puma (N=4), both Bim and Noxa (panel C, N=5) or control siRNA using Amaxa program X-05. Immediately after nucleofection, cells were co-cultured with the CD40L-expressing stroma for 24 hours, followed by incubation with 0.25 or 1 μM MLN4924 or vehicle control for 24 h. Apoptosis within CD19+ subset of cells was determined by Annexin V and 7-AAD staining and normalized to untreated control. Data are the mean ± SE. * - p<0.05 and ** - p<0.01 compared to control siRNA. (D) CLL cells (N=10) were co-cultured with CD40L-expressing stroma for 24 hours, followed by incubation with 0.25 μM MLN4924, 0.1 μM bortezomib, 15 μM U0126, the drugs combined, or with vehicle control for 48 hours. Bortezomib was washed off after 1 hour. Apoptosis was determined as above and normalized to the untreated controls. Data are the mean ± SE. ** - p<0.01 compared to either single drug. For protein detection, cells were incubated with drugs for 24 hours, then lysed and subjected to immunoblotting (representative images out of 3 independent experiments are shown).