Figure 2. TRPV2 expression, plasma membrane targeting, and contribution to basal Ca²⁺ influx, increase with melanoma cell aggressiveness.

1. Analysis for total and plasma membrane (PM) TRPV2 expression in melanoma cell lines. β‐actin was used as loading and cell integrity control. PM fractions were isolated using the surface protein biotinylation technique. The higher molecular weight bands of TRPV2 correspond to the glycosylated and mature channel (Barnhill et al, ²⁰⁰⁴).
2. Constitutive Ca²⁺ influx comparison between resting 501mel (Light blue), WM266.4 (Blue) and 451Lu cells (Dark blue) assessed using the Fura‐2 Mn²⁺ quenching assay. Representative traces of Fura‐2 quenching kinetics are shown on the top panel (each point represents the mean of technical quadruplicates normalized to the baseline). The bar graph summarizes the quantification of the measured quenching rates and is presented as mean ± SEM (n > 3 biological replicates). **P = 0.0032 and ****P < 0.0001, ordinary one‐way ANOVA followed by Dunnett's multiple comparisons post‐test.
3. TRPV2 overexpression in the non‐invasive 501mel cell line (GFP‐TRPV2) or downregulation by lentiviral‐delivery of TRPV2 specific shRNAs (shRNA V2‐1 and V2‐2) in the WM266.4 and 451Lu metastatic melanoma cell lines assessed by western‐blot. β‐actin was used as a loading control.
4. Representative traces of Fura‐2 Mn²⁺ quenching rates in control (GFP) or GFP‐TRPV2 overexpressing 501mel cells, or in control (shRNA Ctrl) or TRPV2 repressed (shRNA V2) WM266.4 and 451Lu cells (each point represents the mean of technical quadruplicates normalized to the baseline).
5. Average normalized quenching rates (as in D) presented as mean ± SEM (n > 3 biological replicates). **P = 0.0095, the Mann–Whitney test (left panel); *P = 0.0399–0.0336 (middle panel) and *P = 0.0438, **P = 0.0033 (right panel) the Kruskal–Wallis test followed by Dunn's multiple comparisons test.

Source data are available online for this figure.