Figure 3.

Quantification of the anti-inflammatory effect induced of RABV on human macrophages and the role of cytoplasmic retention of NF-κB. (A) TNF-α production in human macrophages after 6 h stimulation with LPS (100 ng/mL), with (“+”) or without (“–”) pre-treatment of α-BTX (2 μg/mL), RABV or acetylcholine (Ach; 1 μM), or a combination. Bars represent the mean ± SEM of TNF-α normalized to cells stimulated with LPS only (positive control, dotted horizontal line) for each individual donor (n = 6–9). Horizontal lines represent paired comparisons, whereas asterisks without horizontal lines indicate comparisons with the positive control (LPS only). (B) Nuclear and cytoplasmic NF-κB was quantified by image analysis in ImageJ using a batch image analysis. Bars represent the mean ± SEM of six donors, for which three high-magnification fields were analyzed per treatment. P-values < 0.05 were considered significant and are indicated with an asterisk (*). Representative pictures of NF-κB staining are depicted in panel (C). Pictures where the NF-κB staining (green) completely overlaps with the nuclei (blue) show full nuclear translocation, whereas pictures with non-complete overlap of NF-κB and nuclei indicate cytoplasmic retention of NF-κB. The white scalebar represents 500 μM and all images were acquired at the same magnification.