Inhibition of NF-κB activation in MDS bone marrow samples with BAY11-7082 or bortezomib. (A) Immunofluorescence detection of p65. Bone marrow samples from a high-risk MDS patient (Table 1 no. 54) were precultured for 6 hours in the absence or presence of BAY11-7082 or bortezomib and then subjected to confocal immunofluorescence analyses. Percentages refer to the fraction of cells exhibiting nuclear p65 staining. Similar results were obtained for samples from 2 other high-risk MDS patients (Table 1 nos. 19 and 57). (B-C) BAY11-7082 or bortezomib induced translocation of mitochondrial death effectors in bone marrow cells from high-risk MDS patients. Bone marrow aspirates from the same patients as in panel A were purified to yield CD34⁺ (B) or CD33⁺ (C) cells and cultured ex vivo, in the presence or absence of BAY11-7082 or bortezomib, and then stained with antibodies specific for Hsp60 (red), Cyt c, EndoG, and AIF (all green). Representative cells are shown. Percentage values refer to the cells exhibiting mitochondrial release of Cyt c, EndoG, or AIF in the indicated conditions. Images were acquired as described for Figure 1C.