Figure 1.

TNF treatment induced different sensitivity to cell death in the NF-κB unresponsive NEMO-KO and NEMO-A323P cells. (a) Nemo⁽⁻⁾ MEF reconstituted with empty pBabe-puro vector (KO), wild-type NEMO (NEMO) and IP-associated NEMO-A323P mutant (A323P) were treated for 1 h with 10 ng/ml of TNF. Quantitative real-time PCR was performed in triplicate, and the relative abundance of the indicated transcripts (Icam1 and Nfkbia) was calculated with respect to the expression of Hprt transcript. Error bars represent S.D. of triplicates experiment. (b) KO, NEMO and A323P expressing MEF cells treated with TNF for 6 h. Apoptosis was measured after double staining with Annexin-V and PI. Percentage of Annexin-V/PI double-positive cells and Annexin-V positive, PI-negative cells was shown (as indicated). (c) KO, NEMO and A323P cells were pre-incubated 1 h with DMSO, zVAD (20 mM) or zVAD in combination with Nec1 (30 μM), followed by 6 h of TNF (20 ng/ml) stimulation. Identical concentrations of these death-inducing agents were used in subsequent experiments unless otherwise stated. Relative cell viability was assessed by determining ATP levels with CellTiter-Glo after 6 h of TNF treatment, as a percentage of untreated cells. Data are presented as mean±S.E.M. (three independent experiments performed), *P-value<0.01; **P-value<0.001 (Student's t-test); NS, not significant. Cells were observed on a phase-contrast microscope (right panel). Representative images of three independent experiments are presented. (d) KO, NEMO and NEMO-A323P cells were treated for 2 h with TNF in the presence or absence of zVAD. The luminescence is proportional to Caspase-8 activity, relative light unit (RLU). Error bars represent S.D. of triplicates of one representative experiment of three