Fig. 4

NF-κB is required for hyperphosphorylated tau-mediated cytokine induction. A HT22 cells were transfected with vector or TauP301S following treatment with DMSO or Nec-1 (30 μM) for 48 h, and the lysates were analyzed by western blotting using indicated antibodies. B Representative confocal images (left) and quantification (right) of p65 in HT22 cells transfected with vector or TauP301S following treatment with DMSO or Nec-1 (30 μM) for 48 h. Scale bars, 10 μm. C mRNA was extracted from HT22 cells transfected with vector or TauP301S following treatment with DMSO or TPCA1 (4 μM) and quantified to determine levels of indicated cytokines by qPCR. D Effect of NF-κB inhibitor on the chemotaxis of pTau-induced cytokines on BV2 cells was analyzed by transwell assays, Scale bars, 100 μm. E, F, I HT22 cells were transfected with vector or TauP301S following treatment with DMSO or TPCA1 (4 μM) or TPCA1 (4 μM) + Nec-1 (30 μM) for 48 h; E cell death was measured measuring LDH levels; F levels of the indicated cytokines were analyzed using qPCR; I lysates were analyzed by western blotting using indicated antibodies. G, J HT22 cells were transfected with vector or TauP301S following treatment with DMSO or QNZ (5 μM) or QNZ (5 μM) + Nec-1 (30 μM) for 48 h. G Cell death was evaluated by measuring LDH levels; J lysates were analyzed by western blotting using indicated antibodies. H mRNA was extracted from HT22 cells transfected with vector or TauP301S following treatment with DMSO or SP600125 (5 μM), PH797804 (5 μM), or C176 (2 μM), followed by quantification to determine levels of the indicated cytokines by qPCR. Data are presented as the mean ± standard error of the mean (SEM) of three experiments, statistical analysis was performed using two-tailed unpaired t test in E, G and one-way ANOVA with Dunnett’s multiple comparisons test in C, F. K NC HT22, RIPK1-KO HT22, RIPK3-KO HT22, and MLKL-KO HT22 cells were transfected with vector or TauP301S, and lysates were analyzed by western blotting using indicated antibodies. L Representative confocal images (left) and quantification (right) of p65 in NC HT22, RIPK1-KO HT22, RIPK3-KO HT22, and MLKL-KO HT22 cells transfected with vector or TauP301S. Scale bars, 10 μm. Data are presented as the mean ± standard error of the mean (SEM) of three experiments, and statistical analysis was performed using two-tailed unpaired t test in B, L