Fig. 1.
BANA inhibits NF-κB activation and transcription. (a) A synthesis scheme of (E)-4-(2-nitrovinyl) benzoic acid (BANA, red). (b) BANA inhibits NF-κB activation in NF-κB-RE-Luc transgenic mice. BANA, Benzoic acid (BA), or dimethyl fumarate (DMF) were administered 2 h before intraperitoneal injection of LPS. The graph shows the quantitative analysis in different experimental conditions. All quantitative data are expressed as mean ± SEM; data were analyzed by Ordinary one-way ANOVA followed by Tukey’s multiple comparisons test, ***p = 0.0009 ****p < 0.0001. n = 4 animals per condition. (c) Confocal images of BV2 microglia treated with BANA, BA, or DMF before LPS stimulation. BANA inhibits LPS-induced NF-κB-p65 nuclear translocation. Cells in the sham condition were not treated with LPS. Nuclear NF-κB-p65 colocalizing with DAPI nuclear staining is denoted in yellow. The graph shows the quantitative analysis of the ratio nuclear NF-κB-p65/DAPI among experimental conditions. All quantitative data are expressed as mean ± SEM; data were analyzed by Ordinary one-way ANOVA followed by Tukey’s multiple comparisons test, *p = 0.0148 **p = 0,0031 ****p < 0,0001. 3 replicates of 3 independent experiments. Scale bar = 10 μm. (d) Confocal images show CD11b-positive SOD1G93A primary microglia (gray) treated with BANA, BA or vehicle, before LPS stimulation. BANA inhibits LPS-induced NF-κB-p65 nuclear translocation in SOD1G93A microglia. Nuclear NF-κB-p65 colocalizing with DAPI nuclear staining is denoted in yellow. The graph shows the quantitative analysis of the ratio nuclear NF-κB-p65/DAPI among experimental conditions. All quantitative data are expressed as mean ± SEM; data were analyzed by Ordinary one-way ANOVA followed by Tukey’s multiple comparisons test, ****p < 0.0001, 3 replicates of 4 independent experiments. Scale bar = 20 μm. (e) Analysis by RT-qPCR of mRNA levels of NF-κB-associated proinflammatory cytokines CCL2, IL-6, IL-1β, and TNFα following LPS stimulation in SOD1G93A primary microglia. BANA prevents NF-κB-mediated gene transcription induced by LPS. Data are expressed as mean ± SEM; data were analyzed by Ordinary one-way ANOVA followed by Tukey’s multiple comparisons test, *p = 0,0395 (CCL2) *p = 0.0409 (IL-6) **p = 0.0049 (IL-1β) *p = 0.0292 **p = 0.0063 (TNFα). 2 replicates of 3 independent experiments