Optimization of siRNA treatment in the tested cell lines. A.

<p>The effect of GAPDH knockdown on the proliferative fraction of each cell line was determined. As expected, GAPDH knockdown inhibits proliferation in all cell types. <b>B.</b> Time course of reduction in TRPM8 RNA content by the indicated TRPM8 siRNA treatment. TRPM8 message was determined at the indicated times after transfection. Relative abundance is referred to the amount of mRNA at time 0 and corrected for the expression of transferrin receptor as control. <b>C.</b> TRPM8 protein knockdown by TRPM8.3 and TRPM8.4 siRNA 48 h after transfection. Actin was used as loading control, and the densitometric quantification relative to control siRNA is presented on the right bar diagram.</p

Menthol increases metabolic activity of DU145 cells but does not influence cell cycle distribution. A.

<p>The effect of menthol (two different concentrations) and icilin (10 µM) on the proliferative fraction of each cell line was determined. No effect was detected. Data are presented normalized to the proliferative fraction in the absence of TRPM8 activators. <b>B.</b> In serum-deprived cells, moderate concentrations of menthol elicited an increase in metabolic activity of DU145 cells measured by MTT assay. The effect was weaker at higher concentrations.</p

TRPM8 inhibition impairs migration of prostate tumor cells.

<p>Wound healing assays were performed on PNT1A, LNCaP, PC3 and DU145 cells in the absence or the presence of the TRPM8 inhibitors AMTB and JNJ41876666 (10 µM). A. Representative images of experiments performed in the presence of normal serum concentrations. In the absence of blocker, all cell lines efficiently restore the monolayer after 24 (LNCaP and DU145) or 48 h (PNT1A; at 24 h, no recovery was observed). The presence of blockers impairs healing in LNCaP, PC3 and DU145, but not in PNT1A cells. B. Similar effects were observed on all cell lines already 12 h after the scratch. C. Quantification of restored surface from three images like those in A, for 24 h in all cell lines, except PNT1A (48 h) in the presence of JNJ41876666 (black) and AMTB (red). D. Similar experiments in low serum concentrations revealed that PNT1A did not reduce the scratch surface, while cancer cells LNCaP and PC3 (in 3% FCS) and DU145 (in 1% FCS) did. Healing was again inhibited by JNJ41876666 (10 µM). E. Menthol induced a slight acceleration of healing only in DU145 cells, while icilin showed no effect on any of the lines tested.</p

Pharmacological TRPM8 block affects the cell cycle of prostate cancer cells.

<p>Histograms show changes in the proliferative fraction in the presence of channel blockers as determined by flow cytometry cell cycle analysis. All drugs were used at a concentration of 10 µM. The proliferative fraction under control conditions (vehicle-treated cells) has been considered as 100% of proliferation.</p

Chemical structure of the drugs with TRPM8 antagonist activity used in this study.

<p>Chemical structure of the drugs with TRPM8 antagonist activity used in this study.</p

Expression and functional analysis of TRPM8 in prostate cancer cells.

<p><b>A.</b> mRNA abundance was determined by real time PCR on cDNA derived from RNA from the indicated source. The human transferrin receptor was used as reference housekeeping gene. RNA abundance is expressed as normalized values over PNT1A. Asterisks indicate statistical significance with respect to PNT1A. <b>B–C.</b> DU145 cells respond to cold and menthol<b>. </b><b>B</b>. Transmitted (left) and pseudocolor ratiometric [Ca²⁺]_i images showing an example of the response of DU145 cells to cold (18°C) and menthol 500 µM. <b>C.</b> [Ca²⁺]_i responses of a cold- and menthol-sensitive cell (C1) compared to a cold-insensitive, menthol-insensitive DU145 cell (C2). <b>D–F.</b> Response to cold is diminished by TRPM8 knockdown in DU145 cells. Pseudocolor ratiometric [Ca²⁺]_i images in cells transfected with control siRNA (<b>D</b>) or with TRPM8.4 siRNA (<b>E</b>) at 37°C (upper panels) or 18°C (lower panels). Individual [Ca²⁺]_i responses are represented to the right of the corresponding images. The amplitude and fraction of cells that respond to cold stimuli is clearly diminished in TRPM8 siRNA-treated cultures. This effect is quantitatively depicted in <b>F.</b></p