TRPM8 is endogenously expressed in EC and its stimulation inhibits cell migration. (Ai) Western blot analysis of TRPM8 protein levels in HMECs, HUVECs, and HUAECs. (Aii) Analysis of TRPM8 mRNA expression by qPCR levels and protein expression in HMECs and BTECs. For quantitative PCR, data were normalized to 18S rRNA. Data are mean ± SEM of three different experiments. (B) TRPM8 in HMECs is expressed on the ER. (Bi) Surface biotinylation experiment performed on HMECs and HEK cells overexpressing TRPM8 (HEK-TRPM8 cells). (Bii) Representative confocal images of HMECs showing DAPI staining (blue), calnexin (green), and TRPM8 (red) immunolabeling; the merged image shows the colocalization of endogenous TRPM8 with calnexin, indicating intracellular TRPM8 localization. (C) TRPM8 stimulation inhibits the migration of normal, but not tumor, ECs. (Ci) Representative photographs of a wound-healing assay performed on HMECs treated with control medium, 10 µM icilin, or 40 ng/ml PSA taken at two different time points (t = 0 h, top; t = 8 h, bottom). Bars, 50 µm. (Cii) Plot of the percentage of migration of BTECs (red), HMECs (black), and HUVEC (green) under control conditions (growth medium) or treated with 10 µM icilin. (Ciii) Plot of the percentage of migration of BTECs (B in red) and HMECs (H in black) under control conditions (growth medium) or treated with 40 ng/ml PSA. *, P < 0.05 for comparisons between cells of the same type treated with or without icilin (Cii) or PSA (Ciii).