The proteasome inhibitors ALLN and MG132 down-regulate the constitutive activation of NF-κB and induce apoptosis of CTCL cells. (A) Inhibition of NF-κB DNA-binding activity by ALLN and MG-132 in SeAx cells. Nuclear extracts of SeAx cells were prepared and subjected to EMSA as described in “Patients, materials, and methods.” In one case, cells were either not treated (lanes 1 and 4) or treated with 6.25 μM (lanes 2 and 5) or 12.5 μM (lanes 3 and 6) ALLN during 24 hours or 48 hours, respectively. In the other, cells were not treated (lane 7) or treated with 1 μM MG-132 (lane 8) during the last 5 hours of a culture lasting 48 hours. The data shown here are representative of 2 independent experiments. (B) Increase in annexin-V binding in SeAx cells and CTCL cells from one patient with Sézary syndrome. Cells were treated with or without ALLN (6.25 and 12.5 μM) for 24 and 48 hours or with MG-132 (1 μM during the 5 last hours of culture), as indicated above each panel. Annexin-V binding was carried out with an annexin V–FITC detection kit and analyzed by flow cytometry. Abscissa and vertical axis represent the fluorescent intensities of annexin V–FITC and PI, respectively. For each panel, the numbers in the bottom left, bottom right, and top right quadrants indicate the percentages of annexin V^–/PI^– cells (viable cells), annexin V⁺/PI^– cells (apoptotic, viable cells), and annexin V⁺/PI⁺ cells (dead cells by apoptosis), respectively. Results are representative of 1 of 3 independent experiments.