Fig. 2.
Cytotoxicity of the botulinum C and D proteases. (a) Cell counting assay (CCK-8) was used to monitor neuroblastoma N2A cell viability 40 h after application of the proteases in the presence or absence of Lipofectamine LTX. Botulinum protease type C significantly impaired viability compared with a control condition (*p < 0.01). Application of both proteases with Lipofectamine LTX further lowered cell survival (**p < 0.001). Absorbance at 450 nm (AN450) normalized to control values is proportional to cell count (left panel). The right panel shows the absorbance at 562 nm (AN562) proportional to total protein content in the wells as determined by the bicinchoninic acid (BCA) assay. Botulinum protease type C in the presence of Lipofectamine LTX significantly lowered viability compared with control condition (**p < 0.001) while the addition of the type D protease reduced survival further (*p < 0.01). Results are presented ± SD. (b) Confocal images showing a reduction in number of N2A cells stained with Hoechst 33342. Left panel shows cells treated with Lipofectamine LTX alone, whereas right panel shows cells treated with C and D proteases in the presence of Lipofectamine LTX. White bar: 100 μm. (c) CCK-8 assay was used to monitor viability of the indicated cells following the addition of Lipofectamine LTX with or without botulinum proteases. Control conditions where cells were treated with Lipofectamine LTX alone are shown as white columns. Results were normalized to control (LTX alone, ± SD). A significant (**p < 0.001) loss of viability compared with untreated controls was observed in N2A, SH-SY5Y, Min6, and rat brain cortical cells, which comprise both neurons and non-neuronal cells (*p < 0.01).