Fig. 3: TRPM8 modulators induce melanoma apoptosis.

Representative images from Live/Dead assays in AMM16 (A) and WM266-4 (D) melanoma cells treated for 24 h with TRPM8 modulators 4 and 9 (1 µM). Total cells are stained in green (acridine orange), while dead cells are labeled in red (propidium iodide). Scale bar: 100 µm. Quantification of cell death in AMM16 (B, C) and WM266-4 (E, F) cells treated with compounds 4 and 9 at 1 or 10 µM, respectively, for 24 h. The percentage of dead cells was calculated as: (dead red-stained cells/total green-stained cells) × 100. Representative images of AMM16 (G) and WM266-4 (I) melanoma cells untreated or treated for 6 h with TRPM8 modulators 4 and 9 (1 or 10 μM) and stained with Annexin V–FITC. Contrast phase (CP), Annexin V–FITC, and merged images (overlay) are shown. Scale bar, 100 μm. Quantification of Annexin V–FITC fluorescence in AMM16 (H) and WM266-4 (L) cells untreated or treated as in G, I, measured using a multiwell fluorescence reader (excitation/emission settings optimized for FITC as reported in Methods). Data represent mean ± SD from n independent experiments. Data in B, C, E, F, H, L are presented as mean ± SD from n = 3 independent experiments. Statistical significance was determined. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.