Fig. 6.

Linalool inhibits angiogenesis through reducing intracellular ATP and activating TRPM8. a DCFH fluorescence (in fold of 0 mM linalool) in HDMECs, which were pretreated with or without 0.5 mM DTT, a reducing agent, and then exposed to 0 or 2 mM linalool for 24 h or 0.5 mM H₂O₂ for 30 min, as assessed by flow cytometry (n = 3 independent experiments). b Sprouting (in % of 0 mM linalool) of HDMEC spheroids, which were treated for 24 h with 0 or 2 mM linalool in the presence or absence of 0.5 mM DTT, as assessed by the spheroid sprouting assay (n = 10). c Intracellular ATP level (in % of 0 mM) of HDMECs, which were treated for 24 h with 0 or 2 mM linalool, as assessed by the luciferase assay (n = 4). d Sprouting (in % of 0 mM linalool) of HDMEC spheroids, which were treated for 24 h with 0 or 2 mM linalool in the presence or absence of 1 mM ATP, as assessed by the spheroid sprouting assay (n = 10). e Ca²⁺ signal (F/F₀) in HDMECs, which were pretreated with or without 5 µM AMTB (a specific TRPM8 blocker) for 2 h, loaded with Fluo-4 AM and then stimulated with 0 or 2 mM linalool or 1 mM menthol. The onset of stimulation is indicated by a black arrow. f Peak amplitude (F/F₀) of Ca²⁺ signal in HDMECs, which were pretreated with or without AMTB, loaded with Fluo-4 AM and then stimulated with vehicle, linalool or menthol. g Sprouting (in % of 0 mM linalool) of HDMEC spheroids, which were treated for 24 h with 0 or 2 mM linalool in the presence or absence of 5 µM AMTB, as assessed by the spheroid sprouting assay (n = 10). h Western blot of TRPM8 and β-actin expression in HDMECs, which were transfected for 48 h with 120 nM si-NC or si-TRPM8. i Expression levels of TRPM8/β-actin (in % of si-NC) as assessed by western blot (n = 3 independent experiments). j Sprouting (in % of 0 mM linalool) of HDMEC spheroids consisting of si-NC- or si-TRPM8-transfected cells, which were treated for 24 h with 0 or 2 mM linalool, as assessed by the spheroid sprouting assay (n = 10). Means ± SEM. *P < 0.05 vs. 0 mM linalool or si-NC. ^#P < 0.05 vs. 2 mM linalool. ^§P < 0.05 vs. H₂O₂ or menthol