TRPM8 inhibits EC adhesion through the Rap1–integrin–FAK pathway. (A) TRPM8 acts through the α5β1 integrin signaling pathway. Quantification of adhesion to FN of HMECs treated (or not) with icilin, the integrin-activating 9EG7 antibody both in the presence of 0.1 mM MnCl2. Data represent adhesion normalized to the control untreated condition. (B) Western blot analysis of phosphorylated FAK (pY397 FAK) and total FAK protein levels in HMECs treated or not with 10 µM icilin for 10 min. Quantification of normalized pY397-FAK/FAK protein levels in HMECs from three independent experiments (bottom). (A and B) *, P < 0.05 (Wilcoxon–Mann-Whitney test). (C) Representative images showing anti–β1-integrin antibody 9EG7 (green) and DAPI (blue) in HUVECs stimulated with for 15 or 30 min or untreated cells (t = 0). The bottom panel represents the same images binarized to better underlie the active β1-integrin organization. Bar graph on the right shows quantification of the corrected total cell fluorescence. Data are expressed as mean ± SEM. *, P ≤ 0.05 (two-tailed heteroscedastic Student’s t test).
