Signs of NF-κB activity in P39 cells. (A) NF-κB activation determined by EMSA staining. P39 cells were left untreated (control, Co) or cultured in the presence of BAY11-7082 (5 μM, 6 h), bortezomib (2.5 nM, 6 h), SN50 (36 μM, 6 h), ATRA (1 μM, 48 h), or vitamin D₃ (Vit D₃; 0.25 μM, 48 h), followed by the generation of nuclear extracts and EMSA assays. The transcription factor Oct-1 was monitored to assure equal loading. (B) Detection of p65 by supershift in nuclear extracts of P39 cells. NF-κB was detected by EMSA as in panel A with the difference that a p65-specific antibody was added to the extracts, leading to the apparition of a specific supershifted band. (C) Detection of p65 by confocal immunofluorescence microscopy. Cells treated as in panel A were immobilized on coverslips, fixed, permeabilized, and subjected to immunostaining of p65 (red fluorescence) and nuclear counterstaining (blue), as detailed in “Patients, materials, and methods.” Images were visualized under an LSM 510 confocal microscope equipped with a 63 ×/1.32-0.6 oil-immersion objective lens (Zeiss, Oberkochen, Germany) and a CCD camera (Zeiss). (D) Quantification of immunofluorescence data obtained in panel B. Values are means (X±SD, n = 3). (E) Bortezomib effect on IκB. P39 cells were treated for the indicated period with bortezomib (2.5 nM), followed by immunoblot detection of phosphorylated IκB. (F) Effects of bortezomib and BAY11-7082 on NF-κB target genes. After exposure of cells to either of the 2 NF-κB inhibitors, cell extracts were prepared and the abundance of Bcl-X_L and c-IAP2 was determined. All experiments were repeated at least 3 times, yielding similar results. (G) Lethal knock-down of p65. P39 cells were sham-transfected or transfected with siRNAs that affect the expression of emerin or p65. Immunoblots were performed 72 hours after transfection (bottom panel) and the frequency of annexin-V⁺ cells was monitored by cytofluorometry. Values are means (X±SD, n = 3).