Morphologic alterations and induced cell death after CA-IKK2 expression are NF-κB dependent. (A) Immunoblot of MedB1 and KM-H2 cell transfected with either pRTS-GFP or pRTS-CA-IKK2. Cells were treated with doxycycline (0.5 μg/mL) for 48 hours to induce transgene expression. Cell extracts were analyzed for CA-IKK2 and, as a control, for actin expression. (B) Percentages of dead cells transfected with pRTS-GFP (▭) or pRTS-CA-IKK2 (▬) after induction of transgene expression. Viable and dead cell numbers were determined by trypan blue exclusion. Cells were treated with doxycycline (0.5 μg/m) for 6 days. (C) Immunoblot analysis of Ramos cells stably transduced with IEGZ-empty or IEGZ-TD-IκBα and transfected with inducible pRTS-GFP or pRTS-CA-IKK2. Doxycycline was added to cultures (0.5 μg/mL) for 48 hours to induce transgene expression. (D) Morphologic examination of transfected Ramos cells. Fluorescence microscopy of cells treated with doxycycline for 5 days. Images were taken with a Leica DMIRBE microscope 10× NA = 0.3 PH1 acquired with a Hamamatsu digital camera C4742-95 (Hamamatsu Photonics) and Open Lab software Version 4.0.4 (Improvision). (E) Proliferation assay of Ramos-expressing NF-κB modulators. Ramos cells were stably transduced with IEGZ-empty and transfected with either pRTS-GFP (■) or pRTS-CA-IKK2 (○), or transduced with IEGZ-TD-IκBα transfected with either pRTS-GFP (●) or pRTS-CA-IKK2 (▵). Viable cell numbers were determined by trypan blue exclusion at the indicated time points. (F) Viable cell counts of transduced Ramos cells transfected with either pRTS-GFP (▭) or pRTS-CA-IKK2 (▬). Increased percentages of dead cells after CA-IKK2 expression were reduced in the presence of TD-IκBα. Viable and dead cell numbers were determined by trypan blue exclusion. Cells were treated with doxycycline (0.5 μg/mL) for 6 days. All error bars represent SDs from triplicate experiments.