TRPC3 has a role in PCa cell attraction. (a) PC3 cells were used to study the effect of TRPA1, TRPV2 and TRPC3 on cancer cell attraction. Control HMEC, channel-overexpressing HMEC or hTERT PTEC were plated in multiwell plates, and PC3 cells were plated in the upper chamber of 8 µm-pore culture inserts. hTERT PTEC or TRPC3-overexpressing HMEC (HMEC C3) increased PC3 cells migration through the membrane, quantified 24 h after plating. Each dot indicates the cell number in a single field normalized to PC3 cell count; results are presented as normalized values against co-cultured HMEC, while the continuous line represents the median value of normalized cells/field for each condition (n = 10). The graph shows one representative experiment out of three. Statistical significance: ** p-value < 0.005; *** p-value < 0.0005. Right panel shows the schematic representation of the migration assays used to study PC3 cell migration in co-culture with either hTERT PTEC, TRPA1/V2/C3-overexpressing HMEC, or control HMEC. (b) Chemotaxis chambers were used to study hTERT PTEC-directed migration in the presence of hTERT PTEC-, TRPC3-overexpressing HMEC-, HMEC- or non-conditioned (non-cond) media. Compared to HMEC conditioned medium, PTEC conditioned medium (24-h conditioning) increased PC3 cell speed (c, left panel), while compared to control HMEC conditioned medium, the conditioned medium from TRPC3-overexpressing HMECs significantly promoted PC3 migration via directional persistence index and not on the speed (c, central and right panels). Statistical significance: * p-value < 0.05, ** p-value < 0.005 and *** p-value < 0.0005.