Sensitivity of GalNAc-T-knockout P19 cells to BoNT/C.

<p>(A) Uptake of H_C/C into P19 neurons. P19 neurons were treated with Alexa Fluor 488-labeled H_C/C (green) and NucBlue Live Cell Stain ReadyProbes reagent for nuclear staining (blue), then plasma membranes were stained with CellMask Deep Red Plasma Membrane Stain (red). Differential interference contrast (DIC) and fluorescence images were collected using confocal microscopy. H_C/C was detected as punctate signals in P19 neurons derived from wild-type cells, but not in knockout cells. High degrees of H_C/C and CellMask overlap in merged images are represented in yellow. Scale bars indicate 10 μm. (B) Cleavage of syntaxin-1 and SNAP-25 in P19 neurons by BoNT/C. P19 neurons were exposed to 0.04, 0.16, 0.64, 2.5, or 10 nM BoNT/C for 30 min at 37°C, then the toxin was removed and cells were incubated for 18 h at 37°C in fresh medium. Cells were harvested, solubilized, and extracts were subjected to SDS-PAGE and western blotting. The membranes were probed with anti-GAPDH, anti-syntaxin-1, and anti-SNAP-25 mAbs followed by an HRP-conjugated goat anti-mouse antibody. GAPDH is shown as an internal control. The reactive bands were visualized with SuperSignal West Dura Extended Duration Substrate (upper). The relative amount of intact protein was calculated as a ratio of the relative intensity of each band (intact band/intact + cleaved band). Each point represents the average of three experiments; and because the standard errors for each point were extremely low (<0.01), the error bars are not shown (lower). WT and KO indicate wild-type and GalNAc-T-knockout cells, respectively. Both syntaxin-1 and SNAP-25 were cleaved by BoNT/C in a dose-dependent manner in wild-type cells, but were barely cleaved in knockout cells. (C) Recovery of BoNT/C-induced SNAP-25 cleavage in P19 neurons derived from GalNAc-T-knockout cells by addition of exogenous gangliosides. P19 neurons were treated with the gangliosides GD1a, GD1b, GT1b (each 9 μM), no gangliosides (control), or a mixture of these 3 gangliosides (total 9 μM, each 3 μM) for 24 h. Cells were then exposed to BoNT/C (4 nM) for 30 min. Western blotting analysis was performed at 18 h after removing the toxin for the detection of SNAP-25 cleavage. The relative amount of intact SNAP-25 cleavage was calculated by measuring the intensity of the bands and calculating the ratio of intact to intact + uncleaved bands. Each column represents the average of three independent experiments; error bar indicate the standard error. Significance for differences was evaluated by one-way ANOVA (*, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001).