Figure 4
From: A Novel Inhibitor Prevents the Peripheral Neuroparalysis of Botulinum Neurotoxins
EGA does not inhibit the known steps of BoNT intoxication.
(a) CGNs were treated with EGA (12.5 μM) or vehicle in culture medium at 37 °C. After 30 min, 100 nM cpV-HC/A was added in high K+ buffer for 1 h. Neurons were then washed, fixed and directly imaged. These images are representative of at least three independent sets of experiments. Scale bar, 10 μm. (b) CGNs were treated as in (a) with 250 nM cpV-HC/A and then lysed. The cpV-HC/A content was estimated with a GFP specific antibody. Syntaxin 1A serving as internal control was detected with a specific antibody and an anti-VAMP2 was used to assess BoNT/D cleavage. The amount of cpV-HC/A was determined as a ratio to Syntaxin 1A staining taking the value in non-treated cells (vehicle) as 100%. All data are presented as mean values and error bars indicated the standard deviation obtained from three independent experiments (***p < 0.0001; ns – non significant). (c) CGNs were treated with vehicle or 12.5 μM EGA or 10 nM Bafilomycin A1 for 30 min at 37 °C. Lysotracker Red was then added and the incubation prolonged for further 90 min. Cells were imaged by fluorescence microscopy. The graph shows the quantification of fluorescence intensity of acid compartments (% versus non-treated neurons) arising from CGNs treated with the indicated amount of EGA. Mean and standard deviation values refer to four different experiments. Scale bar, 10 μm. (d) 0.25 μg BoNT/A was reduced in the presence of 12.5 μM EGA for 30 min at 37 °C. 1 μg of GST-SNAP25 was added, the concentration of inhibitor was restored and the reaction was carried out for 12 hrs at 37 °C. SNAP25 cleavage was assessed by SDS-PAGE and Coomassie staining (top-left panel) or immunoblotting (bottom-left panel) with an antibody specific for the BoNT/A-cleaved form of SNAP25. Lane 1 shows untreated GST-SNAP25. Right panel shows the densitometry analysis of western blots, tacking the value of BoNT/A without EGA (vehicle) as 100%. All data are presented as mean values and error bars indicated the standard deviation obtained from three independent experiments.
