Figure 4.

HOIP cleavage dampens NF-κB activation and promotes cell death. A and B, HEK293T cells were transfected with β-gal and NF-κB-luciferase reporter plasmids in combination with the indicated plasmids, normalizing for total DNA. Lysates were harvested 24 h later, and luciferase activity was measured and normalized to β-gal activity. Data are representative of three independent experiments. C, HaCaT cells were transfected with 350 ng of β-gal and NF-κB-luciferase reporter plasmids. 24 h post-transfection, cells were pretreated with 20 μm YVAD for 45 min before stimulation with 75 mJ/cm² UVB. Cells were lysed at the indicated time points post-irradiation, and luciferase activity was measured and normalized to β-gal activity. Data are representative of two independent experiments. D and E, shHOIP HaCaT cells stably expressing either WT or D348E/D387E HOIP were treated for 6 h with the indicated TNF mixtures. RNA was extracted, reverse-transcribed, and subjected to RT-quantitative PCR analysis. Data are representative of two independent experiments and are displayed as mean ± S.E. (error bars). ***, p < 0.001; ****, p < 0.0001. F and G, pLKO.1 or shHOIP HaCaT cells stably expressing either empty vector (EV), WT HOIP, or D348E/D387E HOIP were treated with TNF + CHX for the indicated times, lysed, and subjected to immunoblot analysis (F), or caspase-8 activity was measured (G). Data are representative of two independent experiments and are displayed as mean ± S.E.