Research Article

Development and Characterisation of a Novel NF-κB Reporter Cell Line for Investigation of Neuroinflammation

Figure 3

Proposed endogenous TLR4-ligands can be tested using U251-NF-κB-GFP-Luc cells (a). No changes in NF-κB-dependent luciferase activity were observed using 0.1, 1, 10, or 100 μg/ml fibrinogen. (b) 50 ng/ml high mobility group box 1 (HMGB1) led to a slight decrease whereas vehicle (0.1% cell culture grade BSA in PBS) induced an increase in luciferase activity in U251-NF-κB-GFP-Luc cells. (c) Evaluation of TLR4-dependence in response to HMGB1 using HEK293-MD2-CD14 cells transfected with a transient NF-κB-luciferase reporter and GFP or TLR4-GFP. 50 μM HMGB1 led to a significant decrease in cells transfected with GFP only. E. coli LPS: positive control. Control: nontreated. (d) 5 and 10 μM, but not 0.1 and 1 μM amyloid-β-peptide [1-42], significantly increased NF-κB-dependent luciferase in the U251 reporter cells. (e) HEK293-MD2-CD14 were cotransfected with a transient NF-κB-luciferase reporter and GFP or TLR4-GFP. 5 μM amyloid-β-peptide [1-42], but not heated amyloid-β (control for LPS contamination), significantly increased luciferase activity. E. coli LPS: positive control. Control: nontreated. Data are presented as mean ± SEM from at least 3 independent experiments. , , and were considered significant, ANOVA with Bonferroni correction, CI 95%.
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