Figure 5.

TRPA1 increases EC migration. (a) The ‘prostate-associated’ channels were overexpressed in HMECs to test their effect on cell migration. Expression of TRPA1 increased the migration of HMEC relative to control cells in basal medium, as observed by wound healing experiments. Lines show the mean value ± SEM of one out of three independent experiments (24 h after transfection). Statistical significance **: p-value < 0.005 and ***: p-value < 0.0005 vs. HMEC. (b) Histogram showing the % migration in wound healing experiments of PTEC transfected with control siRNA (siCNTRL) or siRNA against TRPV2 (siTRPV2), under both basal conditions and 8 h after treatment with TRPA1 inhibitor HC030031. Statistical significance **: p-value < 0.005 and ***: p-value < 0.0005 vs. HMECs. (c) TRPA1 downregulation 48 h after siRNA transfection was verified by western blotting. Histogram shows quantification of TRPA1 expression relative to actin represented as the mean + SEM of a minimum of three independent experiments. *: p-value < 0.05. (d) Downregulation of TRPA1 expression was verified 72 h after siRNA transfection in Ca²⁺ imaging experiments by stimulation with 20 µM AITC in control PTEC (siCNTRL, black trace) and in PTECs transfected with siRNA against TRPA1 (siTRPA1, red trace). (e) TRPA1 activity is inhibited when PTEC are treated with a TRPA1 channel inhibitor (20 µM HC030031) based on Ca²⁺ imaging experiments. For experiments in c, d and e traces show the mean value ± SEM of all cells in the recorded field of one representative experiment (left panel); histograms represent the quantification of the peak amplitude after treatment with AITC (c,d) or different doses of the channel inhibitor (10 or 20 µM HC030031) in Ca²⁺ imaging experiments (e, right panel).