Caspase-8 activity is required for modulation of NF-κB activation. (A) Primary monocytes (Mo) were exposed for indicated times (days) to 100 ng/mL M-CSF to trigger their macrophagic differentiation (Mac) or 100 ng/mL GM-CSF plus 10 ng/mL IL-4 for inducing their differentiation into dendritic cells (DC). NF-κB DNA-binding activity was assessed by EMSA (top panel) and the results were analyzed using a PhosphorImager and expressed relative to untreated cells (bottom panel). (B) Monocytes transfected with either luciferase (siLuc) or caspase-8 siRNA (siC8) were treated for 4 days (d4) or 6 days (d6) with M-CSF before quantifying nuclear p65⁺ cells by immunofluorescence. (C-D) U937 cells stably transfected with a caspase-8 dominant-negative mutant (C8-DN) or the corresponding empty vector (Co) were exposed to 20 nM TPA for indicated times (hours). (C) NF-κB DNA-binding activity was assessed by EMSA as in panel A. (D) The percentage of cells with nuclear p65 was determined by immunofluorescence as in panel B. (E-F) U937 cells stably transfected with a lentivirus encoding RIP1 mutated on its caspase cleavage site (RIPm) or the corresponding empty vector (ΔMCS) were exposed to 20 nM TPA for the indicated times (hours). (E) NF-κB DNA-binding activity was assessed by EMSA as in panel A. (F) The percentage of cells with nuclear p65 was determined by immunofluorescence as in panel B; one representative of at least 3 independent experiments, or mean ± SD of 3 independent experiments. * P < .05; ***P < .005.