Noxa up-regulation via transient treatment with bortezomib affects CLL survival. Freshly isolated peripheral blood B-CLL cells were treated for 4 hours with 20 nM proteasome inhibitor bortezomib. Cells were then washed and cultured in fresh medium, or incubation was continued. (A) At the indicated time points, cell lysates were prepared and probed for expression of Noxa, Mcl-1, Bim, and Actin protein by Western blot. Indicated below the lanes: untreated (M), bortezomib washed away after 4 hours (B+), and bortezomib without washing (B-). The decrease in Mcl-1 levels in bortezomib-treated cells at 24 and 48 hours could be inhibited by the pan-caspase inhibitor z-VAD (data not shown). Because of massive cell death after 48 hours in the presence of bortezomib, these lysates did not yield sufficient protein for analysis. (B) Apoptosis of cells was determined by annexin V staining. Spontaneous apoptosis in medium was approximately 50%, which was increased by bortezomib treatment. Results are representative for 3 separate experiments.