Fig. 3. TRPM8 channel activity in RWPE-1 prostate cells.
a Representative images (upper panels) and traces (lower panels) showing [Ca2+]i changes under control solution (CTR) or upon perfusion with menthol (1 mM), WS-12 (1 μM), or icilin (10 μM) on RWPE-1 cells. Time of drugs exposure is indicated by the bar on top of the traces. Quantification of [Ca2+]i peaks measured upon perfusion with TRPM8 activators is reported on the right panel (n = number of analyzed cells). The inset graph indicates the quantification of the total % of cells responsive to tested drugs. b Representative images (left panels) and traces (middle panels) showing [Ca2+]i under control solution (CTR), upon perfusion with menthol (1 mM), WS-12 (1 μM), or icilin (10 μM), or after drugs washout on RWPE-1 M8 cells. Time of drugs exposure is indicated by the bar on top of the traces. Quantification of the [Ca2+]i peaks measured upon perfusion with TRPM8 activators is reported on the right panel (n = number of analyzed cells). The inset graph indicates the percentage of cells responsive to the drugs tested. c Left panel, representative traces of currents evoked by a 100 ms voltage ramp ranging from −100 mV to +100 mV applied every 4 s in control solution (CTL), during application of menthol (500 μM) or after drug washout (W). Right panel, representative time-courses of currents recorded at +80 mV (blue symbols) or −80 mV (red symbols) in single RWPE-1 M8 cells upon exposure to menthol (500 μM). Time of menthol exposure is indicated by the line on top of the traces. d Western blotting analysis with two independent antibodies (D21E4 and 22B1) showing CaMKIIα activation (phosphorylation of Thr286) following WS-12 treatment of RWPE-1 M8 cells. Cells treated with ionomycin were used as positive control for calcium dependent CaMKIIα phosphorylation. Error bars, mean ± SEM. Experiments were performed in at least three experimental sessions; data were analyzed using a two-tailed Student’s t-test. *P ≤ 0.05.