NF-κB activation sensitizes Ramos cells to Fas-mediated apoptosis. (A) Surface expression of Fas on Ramos cells transfected with either pRTS-GFP (top panel) or pRTS-CA-IKK2 (bottom panel). Cells were treated with doxycycline (0.5 μg/mL) for 48 hours, incubated with APO-I antibodies and a corresponding secondary antibody conjugated to Alexa-647 fluorochrome. Stained cells were subjected to flow cytometry and gates were set on GFP⁻ (gray line) and GFP⁺ (black line) fractions. (B) Percentages of dead cells in Ramos transfected with pRTS-GFP or pRTS-CA-IKK2 in the absence (▭) or presence (▬) of agonistic anti-Fas antibody (APO-I). Cells were cultured in the presence of doxycycline for 4 days to induce transgene expression and as indicated treated with APO-I supernatant (5% final concentration [f.c.]) for additional 48 hours. Viable and dead cell numbers were determined by trypan blue exclusion. Error bars represent SDs from triplicate experiments. (C) Fas mRNA expression analyzed by reverse-transcription–PCR. Total RNA of Ramos stably transduced with either IEGZ-empty or IEGZ-TD-IκBα and transfected with pRTS-GFP and pRTS-CA-IKK2, respectively, was isolated 48 hours after addition of doxycycline to the culture medium. (D) Percentages of dead cells in cultures of Ramos-IEGZ-empty (▭) or Ramos-IEGZ-TD-IκBα (▬) transfected with either pRTS-GFP or pRTS-CA-IKK2 after addition of agonistic anti-Fas antibody (APO-I). Fas sensitivity of Ramos-CA-IKK2 is abrogated by TD-IκBα expression. Doxycycline (0.5 μg/mL) was added to the culture medium for 4 days and APO-I supernatant (5% f.c.) was added for additional 24 hours. Viable and dead cell numbers were determined by trypan blue exclusion. Error bars represent SDs from triplicate experiments.