Fig. 2

Effect of botulinum neurotoxin on muscle-nerve coculture function. a Detection by Western blot of cleaved-SNAP25 from hMNs mono-culture (left) or coculture (right) treated with serial doses of rBoNT/A, compared to toxin-free control dose (untreated) using an antibody recognizing cleaved and uncleaved form of SNAP25 protein. b EC₅₀ curve for hMNs in mono-culture or in coculture. DIV 15 hMNs were exposed to rBoNT/A for 24 h before cell lysates were harvested, followed by Western blot to quantify SNAP25 cleavage. c EC₅₀ for hMNs in coculture is 0.49 pM (10^–12.31) and EC₅₀ for hMNs in mono-culture is 0.61 pM (10^–12.21). Data are represented as mean ± SEM (N = 3 independent experiments, each performed in triplicate n = 3). d Effect of rBoNT/A on myotubes contractions 16 hpe (hour post-exposure) and 24 hpe. Coculture was treated at DIV 15 with different doses of rBoNT/A (5 nM, 1 nM, 0.001 nM, 0.00001 nM), and recordings of myotubes contractions were performed before treatment (baseline), 16 h and 24 h after treatment. Data are represented as mean ± SEM (N = 3 independent experiments, each performed in triplicate n = 3). ANOVA with Sidak’s post hoc tests (***p < 0.001; ****p < 0.0001). Significant statistics only 24 hpe, each dose was compared to untreated condition. e Effect of rBoNT/A on myotubes Ca²⁺ oscillations 4 hpe and 7 hpe. Cells were stained with 2 µM Cal520 dye the day of the recording. Coculture was treated at DIV 15 with different doses of rBoNT/A (5 nM, 1 nM, 0.001 nM, 0.00001 nM), and recordings of Ca²⁺ oscillations were performed before treatment (baseline), 4 h and 7 h after treatment. d-e Data are represented as mean ± SEM (N = 3 independent experiments, each performed in triplicate n = 3). ANOVA with Sidak’s post hoc tests (^##p < 0.01; ^####,****p < 0.0001). Significant statistics 4 hpe (represented by ^#) and 7 hpe (represented by *), each dose was compared to untreated condition