Fig. 2

Silencing CYLD did not abolish the NF-κB pathway. a HT-22 cells were transfected with control or CYLD siRNA and a luciferase-driven NF-κB reporter plasmid. HT-22 cells were treated with glutamate (5 mM, 8 h) as indicated. Luminometric detection revealed a similar NF-κB activity in CYLD-depleted cells as compared to controls. Mean values and S.D. of n = 6 experiments per group are shown. b HT-22 cells were transfected with control or CYLD siRNA and NF-κB reporter plasmid for 24 h, and the cells were treated with TNF-α (100 ng/ml, 8 h) as indicated. Mean values and S.D. of n = 4 experiments per group are shown; ***p < 0.001, compared to untreated control (ANOVA, Scheffe's). c Western blot analysis of IκB and P-IκB levels in CYLD-depleted cells, treated with or without glutamate (5 mM, 8 h). Treatment with TNF-α (100 ng/ml, 1 h) was carried out as a positive control. NF-κB remained constitutively inactive, regardless of CYLD depletion. d MTT cell viability assay showing exogenous addition of TNF-α (100 ng/ml) did not enhance glutamate 5 mM (10 h) toxicity in HT-22 cells