Fig. S5. Involvement of vesicular exocytosis in mechanically induced ATP-dependent paracrine signalling in MCC13 cells.

Fig. S5. Involvement of vesicular exocytosis in mechanically induced ATP-dependent paracrine signalling in MCC13 cells. (a) Representative [Ca²⁺]ᵢ traces in MCC13 cells following mechanical stimulation (red arrows) under control conditions (left panel) and after treatment with bafilomycin A1 (Baf.), a vesicular exocytosis inhibitor, together with AG1478, an EGFR tyrosine kinase inhibitor (right panel). [Ca²⁺]ᵢ was measured by live-cell ratiometric fluorescence imaging using Fura-2 AM. (b) Summary scatter plot for (a). The scatter plot (left) summarizes the [Ca²⁺]ᵢ responses of the mechanically stimulated cells (red dots) and neighbouring cells (blue dots) under control and Baf. plus AG1478-treated conditions. Each dot represents data from an independent experiment (n = 5 for control, n = 9 for Baf. plus AG1478), with neighbouring cell responses averaged from up to 6 cells per experiment. Data are represented as mean ± SEM. Statistical analysis showed NS for mechanically stimulated cells (unpaired t-test) and P < 0.001 vs. control for neighbouring cells (Mann–Whitney test). The stacked bar graphs (right) summarize the number of responsive cells among the neighbouring MCC13 cells under the first (control, total of 112 cells) and second (Baf. plus AG1478-treated, total of 167 cells) stimulations. (c) Representative time-dependent traces of [ATP]ᵢ in MCC13 cells following mechanical stimulation (red arrow) under control conditions (upper) and Baf. plus AG1478-treated conditions (lower). Intracellular ATP was monitored using the fluorescent ATP indicator ATP-Red 1 and quantified as F/F₀. (d) Summary scatter plot for (c), comparing the mechanical stimulation-induced [ATP]ᵢ decrease between control and Baf. plus AG1478-treated conditions. Each dot represents the average reduction in [ATP]ᵢ from baseline to indentation in an independent experiment (n = 6 for control, n = 9 for Baf. plus AG1478). Data are represented as mean ± SEM. Statistical analysis showed P < 0.01 (Mann–Whitney test). Inhibition of vesicular exocytosis with Baf. plus AG1478 attenuated indentation-evoked Ca²⁺ increases in neighbouring cells and reduced the associated decrease in [ATP]i levels, indicating exocytosis-dependent ATP release.Supplementary figure associated with: Seo MS et al. Merkel cells release ATP in response to mechanical stimulation to mediate paracrine signalling. British Journal of Dermatology (2026). https://doi.org/10.1093/bjd/ljag067