Induction of apoptosis by NF-κB inhibition in P39 cells. (A-F) Kinetics of apoptosis induction by different doses of BAY11-7082 (A-C) and bortezomib (D-F). P39 cells were cultured in the presence of either of 2 NF-κB inhibitors for the indicated period and then stained with DiOC₆(3) (A,D), FITC-labeled annexin-V (B,E), and/or PI (C,F) to determine the frequency of ΔΨ_m^low, phosphatidylserine⁺, and dead (PI⁺) cells, respectively. (G-I) Effect of z-VAD-fmk on apoptotic characteristics of P39 cells dying upon NF-κB inhibition. P39 cells were cultured for 24 hours in the absence or presence of BAY11-7082 (5 μM), bortezomib (2.5 nM), or z-VAD-fmk (100 μM), followed by staining with DiOC₆(3) plus PI (G), FITC-labeled annexin-V (AnnV) plus PI (H), or Hoechst 33324 (I) to determine the ΔΨ_m loss (G), phosphatidylserine exposure (H), cell death (G-H), or karyorrhexis (I), using either cytofluorometry (G-H) or fluorescence microscopy (I). Image was visualized using a Leica epifluorescent microscope equipped with a 63 ×/1.32-0.6 oil-immersion objective lens and CCD camera (Leica, Heidleberg, Germany). Values are means ± SD (n = 3). (J-K) Immunoblot detection of activated caspase-3 in P39 cells treated with the NF-κB inhibitors BAY11-7082 (J) or bortezomib (K), alone or in combination with z-VAD-fmk. The concentrations of the agents were the same as in panels G-I, and the incubation period was 12 and 24 hours.